KR101194443B1 - Composition for promoting bone formation - Google Patents

Abstract

The present invention is a composition for promoting bone formation, orthodontic composition and osteoporosis, fracture, bone formation disorder, or malocclusion treatment (improved) containing 20 (S)-ginsenoside Rh2 as an active ingredient (improvement) Or a prophylactic composition.
The compositions of the present invention have the advantage of being effective in promoting bone formation, orthodontics, and in the treatment (improvement) or prevention of one or more bone related diseases selected from osteoporosis, fractures, bone formation disorders, or malocclusion.

Description

Composition for promoting bone formation

The present invention relates to a composition for promoting bone formation, preferably a pharmaceutical composition for promoting bone formation by promoting bone formation, food composition, orthodontic pharmaceutical composition, food composition and pharmaceutical composition for treating or preventing bone-related diseases, It relates to a food composition for improvement or prevention.

The bone of the human body maintains its homeostasis through the process of bone modeling and bone remodeling. Bone formation begins in the early stages and lasts until the young adult period when the skeleton matures and the growth ends. Osteoblast activity is greater than bone resorption due to the activity of osteoclasts. However, when bone absorption increases due to various causes as age increases, bone tissue becomes vulnerable and causes various bone diseases such as osteoporosis and fractures. Thus, there is a need to develop bone formation promoters to maintain homeostasis.

Bone-related diseases include osteoporosis, fractures, bone formation disorders, malocclusion, and the like. Osteoporosis is a skeletal disease that increases the risk of fractures and bone fragility due to low bone marrow and destruction of the bone matrix. Done. Osteoporosis is a condition in which bone constituents (particularly calcium) and substrates are reduced, and osteoclasts are increased than osteoclasts due to the balance of aggregate formation. The normal bone inside has a dense structure like a net, but in the case of osteoporosis, the gap between the structures is widened and the microstructure is thinned and weakened, so that the bone can be easily broken even in the small impact.

In osteoporosis, bisphosphonate-based therapies such as Fosamax (alendronate) and Actonel (risedronate) are used, but most of these bisphosphonate agents weaken and kill osteoclasts that destroy bone. Induces slowing or stopping bone loss. However, these drugs do not have a function of promoting the formation of new bones, so the development of drugs or foods that act to promote the formation of bone is necessary.

Fracture refers to the fracture of bone with abnormally broken continuity of bone, bone plate, or joint surface. The causes of the fracture include trauma such as traffic accidents, safety accidents in the workplace, bone changes caused by diseases such as osteoporosis, bone cancer, metabolic disorders, and stress on the bones repeatedly caused by sports or loads.

Osteodystrophy, also known as osteotrophy, is a disease of bone caused by chronic kidney failure. It is caused by congenital abnormal kidney function and dies when the kidneys are weak and not brittle. This bone disease is called Renal Osteodystrophy. Bone diseases related to osteotrophy include osteomalacia and osteotease fivrosa.

On the other hand, orthodontic treatment is performed to treat or prevent irregular dentition, such as malocclusion. Orthodontic tooth movement for orthodontics involves turnover and changes in physiological alveolar bone replacement by orthodontic forces. During orthodontic tooth movement, alveolar bone replacement process involves bone formation on the tension side and bone absorption on the compression side. Bone absorption occurs early (3-5 days) during the tooth movement, and then reverse and later (7-14 days). It is known that bone formation occurs not only in the alveolar bone but also throughout the periodontal tissue. Bone resorption induced by orthodontic power lasts for 1-14 days, and osteoclasts derived from bone marrow stem cells appear after 12 hours on the compression side during tooth movement and exist for 7 days. Acid phosphatase (ACP) and tartrate-resistant acid phosphatase (TRAP) It is known that high activity, bone formation-related cells show high alkaline phosphatase (ALP) activity and orthodontic bone formation lasts up to 21 days. Since orthodontic correction can be performed quickly and effectively by orthodontic force promoting bone formation by orthodontic force, it is necessary to develop a drug or food that can be orthodontized by promoting bone formation.

On the other hand, 20 (S) -ginsenoside Rh2 is not known with respect to promoting bone formation.

One technical problem to be solved by the present invention is to provide a composition for promoting bone formation.

Another technical problem to be solved by the present invention is to provide a composition for orthodontics.

Another technical problem to be solved by the present invention is to provide a composition for the treatment (improvement) or prevention of one or more bone-related diseases selected from osteoporosis, fracture, bone formation disorder, malocclusion.

The present invention provides a pharmaceutical composition for promoting bone formation containing 20 (S) -ginsenoside Rh2 as an active ingredient.

The 20 (S) -ginsenoside Rh2 may be represented by the following formula (I).

<Formula I>

The 20 (S) -ginsenoside Rh2 may be prepared or purchased by a known method.

The 20 (S) -ginsenoside Rh2 may be intact or in the form of a pharmaceutically acceptable salt, and is not particularly limited as long as it is a pharmaceutically acceptable salt. For example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, Salts such as hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, or naphthalenesulfonic acid.

The bone formation refers to the formation of bone tissue by the deposition of lime on the bone tissue, may be due to one or more selected from the induction of differentiation or calcification of osteoblasts.

By promoting bone formation, it is possible to treat or prevent one or more bone-related diseases selected from osteoporosis, fracture, bone formation disorder, or malocclusion.

The present invention also provides a pharmaceutical composition for orthodontics containing 20 (S) -ginsenoside Rh2 as an active ingredient. Orthodontics means to correct irregular teeth. The composition may be for promoting tooth movement that exhibits orthodontic effect by promoting tooth movement, and the tooth movement promoting may be due to bone formation promotion during orthodontic tooth movement. Preferably, by promoting bone formation on the tension side during orthodontic tooth movement, bone formation by orthodontic force is possible in the alveolar bone and the entire periodontal tissue.

The present invention also provides a pharmaceutical composition for treating or preventing one or more bone-related diseases selected from osteoporosis, fracture, bone formation disorder, or malocclusion containing 20 (S) -ginsenoside Rh2 as an active ingredient.

The treatment or prevention may be by promoting bone formation (see Korean Society for Fractures Vol. 23, No. 3, 1225-1682, Medical Information Vol. 35, No. 4, No. 406, 25-35, Korean Journal of Internal Medicine, Vol. 470, 477-481). .

In addition, the present invention provides a food composition for promoting bone formation containing 20 (S) -ginsenoside Rh2 as an active ingredient.

In addition, the present invention provides a food composition for orthodontics containing 20 (S) -ginsenoside Rh2 as an active ingredient.

In addition, the present invention provides a food composition for improving or preventing one or more bone-related diseases selected from osteoporosis, fracture, bone formation disorder, or malocclusion, containing 20 (S) -ginsenoside Rh2 as an active ingredient.

In the food composition of the present invention, the same applies as long as there is no contradiction mentioned in the pharmaceutical composition of the present invention.

In the present invention, the food composition may be a health functional food, the formulation is prepared according to a conventional method, and may be encapsulated after drying with a carrier or in the form of other tablets, granules, powders, beverages, porridge and the like. In addition to the above, it can be produced in any food form.

The active ingredient contained in the composition of the present invention exhibits an osteogenic effect, orthodontic effect, osteoporosis, fracture, bone formation disorder, or at least one bone related disease treatment (improvement) or prevention effect.

Accordingly, the present invention is directed to the treatment (improvement) or prevention of one or more bone-related diseases selected from the use of promoting active bone formation, orthodontic use, osteoporosis, fracture, bone formation disorder, or malocclusion of the active ingredient contained in the composition of the present invention to provide.

In addition, the present invention is one selected from the method of promoting bone formation, orthodontics, osteoporosis, fracture, bone formation disorder, or malocclusion, comprising administering to the mammal (including human) the active ingredient contained in the composition of the present invention. Provided are treatments (improvements) or preventive methods for abnormal bone related diseases.

Unless otherwise stated, the content of the composition of the present invention is equally applicable to the uses and methods of the present invention.

The active ingredient in the composition of the present invention can be administered orally or parenterally to mammals including humans, and the active ingredient can be formulated in combination with a pharmaceutically acceptable carrier. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and / or surfactants can be used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose, and And / or by adding gelatin or the like. Lubricants such as magnesium and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions and syrups, and various excipients such as wetting agents, sweeteners, fragrances and / or preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. May be included. Formulations for parenteral administration include injectable solutions, suspensions, emulsions, lyophilizers, nasal washes, or suppositories. Injectable solutions, suspensions and emulsions can be prepared by mixing water, non-aqueous solvents or suspending solvents with the active ingredient. As non-aqueous solvents and suspending solvents, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and ethyl oleate Injectable esters such as and the like. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol and / or gelatin may be used. The pharmaceutical composition of the present invention can be administered by subcutaneous injection, intravenous injection or intramuscular injection during parenteral administration.

In the compositions, uses and methods of the present invention, it can be used in a dose of 5 to 10 mg / kg per day on an adult basis.

The active ingredient may be formulated by adding a pharmaceutically or food-acceptable carrier, excipient or diluent, etc., and the formulation may be found in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA et al. Reference may be made.

The active ingredient in the composition of the present invention may be included in 40 to 80% by weight, preferably 50 to 80% with respect to the total weight.

The composition of the present invention is effective for promoting bone formation, orthodontics, and for treating (improvement) or preventing one or more bone-related diseases selected from osteoporosis, fractures, bone formation disorders, or malocclusion.

1 is a result of ALP activity measurement and calcification (A) and Von Kossa and Alizarin Red staining results (B) to confirm the effect of promoting the formation of bone by 20 (S) -ginsenoside Rh2.
Figure 2 shows the results of confirming changes in bone differentiation gene expression by 20 (S) -ginsenoside Rh2.
Figure 3 Western blot results (A ~ C) confirming the change in PKD activity by 20 (S)-ginsenoside Rh2, bone differentiation promoter expression change results (D) and ALP activity measurement and calcification activity measurement results (E ).
Figure 4 shows the Western blot results (A ~ D) and ALP activity measurement and calcification measurement results (E) confirming the change in p38 activity by 20 (S) -ginsenoside Rh2.
Figure 5 shows the Western blot results (A ~ C, E) and ALP activity measurement and petrification measurement results (D) confirming the change in AMPK and ACC activity by 20 (S)-ginsenoside Rh2.

Hereinafter, the present invention will be described in more detail with reference to Examples and Preparation Examples, but the following Examples and Preparation Examples are only for illustrating the present invention, and the content of the present invention is not limited to the following Examples or Preparation Examples.

T-tests on the experimental results of the following examples were performed to determine statistical significance when the difference was less than 5% (p <0.05) or less than 1% (p <0.01).

Example 1 Confirmation of Bone Formation Promoting Effect and Orthodontic Effect of 20 (S) -ginsenoside Rh2

1-1. Preparation of 20 (S) -ginsenoside Rh2

20 (S) -ginsenoside Rh2 was prepared by purchasing from the Embossing Research Institute (Hongin Tower 1511, 536-9 Bongmyeong-dong, Yuseong-gu, Daejeon, Korea).

The prepared 20 (S) -ginsenoside Rh2 was used by dissolving in 0.1% DMSO (dimethylsulfoxide) when treating the sample.

1-2. Preparation and Processing of Cells

The MC3T3-E1 cell line (Riken Cell Line Bank), known as osteoblasts, was prepared with Dulbecco's modified Eagle's medium (DMEM) medium (Gibco BRL, USA) supplemented with 10% fetal calf serum and 1% penicillin / streptomycin (Invitrogen Corporation, CA, USA). ) Were passaged every 3 days in a 37 ° C., 5% CO ₂ incubator. 6-well plates were then inoculated with ⁵ × 10 ⁵ cell numbers, respectively, followed by 50 ug / ml ascorbic acid (Sigma-Aldrich Inc., MO, USA) and 10 mM beta-glycerophosphate (Sigma-Aldrich Inc., MO, USA). ) Differentiation was induced for 14 days in DMEM medium.

1-3. Experiment for measuring changes in gene expression involved in differentiation promotion

Total RNA in the samples (cells) was isolated using the TRIzol reagent (Invttrogen Corporation, CA, USA) according to the manufacturer's instructions. Specifically, the cultured cells were washed twice with cold PBS and then lysed with 1 ml of TRIzol reagent. After shaking 200μl of chloroform and centrifuged at 12000rpm at 4 degrees Celsius, the supernatant was separated. The same amount of isopropanol was added to the separated supernatant, shaken, and then centrifuged again at 12000 rpm at 4 degrees Celsius. The supernatant was discarded and the remaining pallet was washed twice with 70% ethanol and RNA was isolated. The isolated RNA was quantified and confirmed by absorbance measurement at 260 nm. 5 μg of the isolated total RNA was added to the reagent in the RT-PCR amplification kit and reacted for 60 minutes at 45 degrees Celsius, and the reaction was terminated at 4 degrees Celsius for 10 minutes to prepare cDNA. cDNA was amplified by the PCR method using the primers of Table 1. PCR reactions were carried out using SYBR Green reagent (Roche, Basel, Switzerland) according to the manufacturer's instructions. Specifically, 25 μl of SYBR green was added to the same amount of cDNA, and then amplified by reacting for 30 seconds at 95 degrees Celsius and 1 hour at 50 degrees Celsius.

[Table 1]

In the above table, HPRT is a control gene required for quantitative analysis.

1-4. Alkaline phosphatase (ALP) activity measurement experiment

ALP is an enzyme that is an indicator of bone formation and is expressed in the early and mid-cell differentiation. Samples (cells) were suspended in 0.1% Triton X-100 and lysed by an ultrasound machine (Branson, USA). The lysed cells were centrifuged (12000 g, 4 degrees Celsius), the supernatant was quantified by the Bradford method, and the same amount of protein was purified using ALP kit (Sigma-Aldrich co. MO, USA) according to the manufacturer's instructions. Was measured. Specifically, the cultured cells were washed with cold PBS buffer, and then 0.1% Triton-X solution was added to recover the cells. The recovered cells were sonicated to break the cell membrane, and then centrifuged at 4 ° C. and 12000 rpm for 20 minutes. The supernatant was collected and quantified in the same amount, and then the reagent was added to the ALP kit to measure the absorbance at 405 nm.

1-5. Mineralization Measurement Experiment

Calcium accumulated extracellularly during bone formation was confirmed by staining with Alizarin Red S (Sigma-Aldrich Co., MO, USA) and VonKossa (Invitrogen Corporation, CA, USA). Samples (cells) were fixed with 10% formalin for 1 hour, and then stained for 10 minutes with 1% Alizarin Red solution or 1% Von Kossa solution.

1-6. Protein expression measurement experiment

Western blotting was performed to confirm the expression changes of the signal transduction mediators related to cell differentiation.

Samples (cells) were homogenized with lysis buffer for protein analysis. Protein quantification was measured using a Bio Rad assay reagent (Bio-Rad, USA), and 20 μg of the quantified protein was isolated by 8% SDS-PAGE. Afterwards, the gel was transferred to a membrane (Milipore, Cat.No: IPVH00010), blocked with 5% skim milk for 1 hour at room temperature, and diluted with a 1: 1000 ratio of primary antibody (PPKD, Cell Signaling, 2054; PKD, Cell Signaling 2052). ; PP38, Cell Signaling, 9212; phospho-p38; Cell Signaling, 4631; P-AMPK, Cell Signaling, 2531; AMPK, Cell Signaling, 2532; P-ACC, Cell Signaling, 3661; ACC, Cell Signaling, 3662; Actin , Santacruz, sc-1616) and reacted overnight at 4 ℃. After washing three times with tris-buffered saline tween-20 (TBST), the mixture was reacted with secondary antibody (Santa Cruz, sc-2313) diluted at a ratio of 2000 to 1 hour at room temperature. After washing 3 times with TBST and using the ECL solution (Amersham, Sweden) was developed on the X-ray film.

1-7. Confirmation of bone formation promoting effect of 20 (S) -ginsenoside Rh2

The cells prepared in the same manner as in 1-2 were treated with 20 (S) -ginsenoside Rh2 (0, 10, 20, 40 uM) at the onset of differentiation induction, and the experiment of 1-4. At 7 days of differentiation induction. ALP activity was measured according to the method. The results are shown in FIG. 1A. Fold increase means the relative ratio of 1 to the control (20 (S) -ginsenoside Rh2 untreated group).

In addition, 20 (S) -ginsenoside Rh2 (0, 10, 20, 40 uM) was treated in the same manner as the ALP activity measurement, and Alizarin was petrified according to the experimental method of 1-5. Red staining and von Kossa staining were used. The results are shown in FIG. 1B and Alizarin red (Sigma-Aldrich. Inc. MO, USA) staining was quantified by destaining with ethylpyridium chloride (Sigma-Aldrich. Inc. MO, USA) and measuring the absorbance at 550 nm. It is shown graphically in Figure 1A.

As a result, it can be seen that 20 (S) -ginsenoside Rh2 increases ALP activity in a concentration-dependent manner. In addition, Alizarin Red staining and Von Kossa staining confirmed the accumulation of calcium, indicating that 20 (S) -ginsenoside Rh2 increases calcium accumulation in a concentration-dependent manner.

Therefore, it was confirmed that the bone formation is promoted by 20 (S) -ginsenoside Rh2, and as a result, it can be seen that the bone formation promoting effect is effective in osteoporosis, fracture, bone formation disorder, malocclusion and the like. In addition, fast and effective orthodontic treatment is possible by the bone formation promoting action of 20 (S) -ginsenoside Rh2 during orthodontic tooth movement. In other words, by promoting bone formation on the tension side during orthodontic tooth movement, bone formation by orthodontic force is possible in the alveolar bone and the entire periodontal tissue. In particular, the effect can be further increased by selectively applying (coating) 20 (S) -ginsenoside Rh2 to the tension side.

1-8. Confirmation of Gene Expression Changes Promoting Bone Formation Differentiation of 20 (S) -ginsenoside Rh2

Differentiation induction was 0, 3, for the group treated with 20 (S) -ginsenoside Rh2 (40 uM) and the untreated group (-) at the onset of differentiation induction for cells prepared in the same manner as in 1-2. MRNA expression levels of bone formation differentiation promoting genes {ALP, OCN (osteocalcin), OPN (osteopontin), Osx (osterix), Col-I (collagen type I)} Was measured. The results are shown in FIG. 2A. In the figure, a fold increase means a relative ratio where the mRNA expression level at the start of differentiation induction (day 0 of differentiation induction) is set to 1. As a result, the expression of ALP, which is known to be expressed at the beginning of differentiation, was increased on the third day of induction, and the expression of OCN, OPN, Osx, Col-I, which is known to be expressed after the middle of differentiation, was increased after 7 days of differentiation. It was.

In addition, the cells prepared in the same manner as in 1-2. Were treated with 20 (S) -ginsenoside Rh2 (0, 10, 20, 40 uM) at the onset of differentiation induction, and then differentiated induction at 1-3 days. MRNA expression levels of bone formation differentiation promoting genes (ALP, OCN, OPN, Osx, Col-I) were measured according to the experimental method. The results are shown in FIG. 2B. In the figure, a fold increase means a relative ratio where the mRNA expression level at the start of differentiation induction (day 0 of differentiation induction) is set to 1. As a result, it can be seen that 20 (S) -ginsenoside Rh2 increases the expression of bone formation differentiation promoting genes in a concentration-dependent manner.

The same method as in 1-3, except that the forward sequence (5'-GCCGGGAATGATGAGAACTA-3 '; SEQ ID NO: 13) and the reverse sequence (5'-GGACCGTCCACTGTCACTTT-3'; SEQ ID NO: 14) as the primer sequence in the experiment In addition, mRNA expression for Runx2, an early important gene of bone formation, was also measured. 2A and B, there was no change in the expression of Runx2, but it was confirmed that Runx2 increased the transactivity by 20 (S) -ginsenoside Rh2 from the increase in the expression of Osx, which is a lower signal of Runx2.

From these results, it was confirmed that the bone formation differentiation promoting gene expression is promoted by 20 (S) -ginsenoside Rh2 to promote bone formation. As a result, osteoporosis, fracture, bone formation disorder, It can be seen that it is effective for malocclusion. In addition, fast and effective orthodontic treatment is possible by the bone formation promoting action of 20 (S) -ginsenoside Rh2 during orthodontic tooth movement. In other words, by promoting bone formation on the tension side during orthodontic tooth movement, bone formation by orthodontic force is possible in the alveolar bone and the entire periodontal tissue. In particular, the effect can be further increased by selectively applying 20 (S) -ginsenoside Rh2 on the tension side.

1-9. Confirmation of PKD (Protein Kinase D) Activation Effect of 20 (S) -ginsenoside Rh2

To investigate the effect of 20 (S) -ginsenoside Rh2 on the activation of PKD, we tried to confirm the bone formation mechanism of 20 (S) -ginsenoside Rh2.

The cells prepared in the same manner as in 1-2 were treated with 20 (S) -ginsenoside Rh2 (40 uM) at the onset of differentiation induction, followed by experiments of 1-6. At 0, 15, 30 and 60 minutes of differentiation induction. Western blot was performed according to the method to confirm the expression changes of pPKD and PKD.

The results are shown in FIG. 3A. In the figure, pPKD means the amount of phosphorylated PKD protein, PKD means the amount of PKD protein of the base level. Actin refers to a control protein as a loading control. As shown in Figure 20 (S)-ginsenoside Rh2 it can be seen that activates PKD in a time-dependent manner.

In addition, the cells prepared in the same manner as in 1-2. Were treated with 20 (S) -ginsenoside Rh2 (0, 10, 20, 40 uM) at the start of differentiation induction, and then 1-6. Western blot was carried out according to the experimental method of to determine the expression changes of pPKD and PKD.

The results are shown in FIG. 3B. In the figure, pPKD means the amount of phosphorylated PKD protein, PKD means the amount of PKD protein of the base level. Actin refers to a control protein as a loading control. As shown in the figure, 20 (S) -ginsenoside Rh2 can be seen to activate PKD in a concentration-dependent manner.

In addition, PKD activity inhibitors were applied to the cells prepared in the same manner as described in 1-2. For the group treated with 20 (S) -ginsenoside Rh2 (40 uM) and the untreated group (-) at the onset of differentiation induction. After treatment with Go6976 (Calbiochem, CA, USA) (10, 20uM) or untreated (-), Western blot was performed at 60 minutes of differentiation induction according to the experimental method of 1-6. To change the expression level of pPKD and PKD. Confirmed.

The results are shown in Figure 3C. In the figure, pPKD means the amount of phosphorylated PKD protein, PKD means the amount of PKD protein of the base level. Actin refers to a control protein as a loading control. As shown in the figure, when both 20 (S) -ginsenoside Rh2 and Go6976 were not treated (-,-) or only Go6976 was treated (20uM), PKD was not phosphorylated and was not activated, and 20 (S) -When only ginsenoside Rh2 was treated, PKD was phosphorylated and activated, but when both 20 (S) -ginsenoside Rh2 and Go6976 were treated, it was found that PKD activity was inhibited in a concentration-dependent manner on Go6976.

In addition, PKD activity inhibitors were applied to the cells prepared in the same manner as described in 1-2. After treatment (10, 20uM) or untreated (-) of Go6976, mRNA of osteogenic differentiation promoting genes (ALP, OCN, OPN, Osx, Col-I) according to the experimental method of 1-3. Expression level was measured. The results are shown in FIG. 3D. The fold increase in the figure means a relative ratio of the mRNA expression level of 20 (S) -ginsenoside Rh2 and Go6976 untreated. As a result, it was confirmed that the expression of ALP, OCN, OPN, Osx, and Col-I increased by 20 (S) -ginsenoside Rh2 was suppressed in Go6976 in a concentration-dependent manner.

In addition, PKD activity inhibitors were applied to the cells prepared in the same manner as described in 1-2. After Go6976 was treated (10, 20 uM) or untreated (-), ALP activity was measured according to the experimental method of 1-4. The results are shown in FIG. 3E. Fold increase means the relative ratio of the mRNA expression level of 20 (S) -ginsenoside Rh2 and Go6976 untreated as 1.

In addition, PKD activity inhibitors were applied to the cells prepared in the same manner as described in 1-2. After Go6976 was treated (10, 20 uM) or untreated (-), the degree of petrification was measured according to the experimental method of 1-5. After staining the degree of petrification through Alizarin Red staining and destaining was numerically measured by the method of measuring the absorbance is shown graphically in Figure 3E. Fold increase means the relative ratio of the mRNA expression level of 20 (S) -ginsenoside Rh2 and Go6976 untreated as 1.

As shown in FIG. 3E, it was confirmed that increased ALP activity and mineralization by 20 (S) -ginsenoside Rh2 were inhibited concentration-dependently in Go6976.

From these results, it was confirmed that 20 (S) -ginsenoside Rh2 promotes activation of PKD, an important signal substance for initiating bone formation, and promotes bone formation. As a result, osteoporosis, fracture, It can be seen that it is effective in bone formation disorder, malocclusion, and the like. In addition, fast and effective orthodontic treatment is possible by the bone formation promoting action of 20 (S) -ginsenoside Rh2 during orthodontic tooth movement. In other words, by promoting bone formation on the tension side during orthodontic tooth movement, bone formation by orthodontic force is possible in the alveolar bone and the entire periodontal tissue. In particular, the effect can be further increased by selectively applying 20 (S) -ginsenoside Rh2 on the tension side.

1-10. 20 (S) -ginsenoside Rh2 confirmed the effect of increasing p38 activity

To determine the effect of 20 (S) -ginsenoside Rh2 on the increased activity of p38, the lower signaling factor of PKD, the following method to identify the signaling mechanism promoting bone formation by 20 (S) -ginsenoside Rh2 Experiment with.

The cells prepared in the same manner as in 1-2 were treated with 20 (S) -ginsenoside Rh2 (40 uM) at the onset of differentiation induction, followed by experiments of 1-6. At 0, 15, 30 and 60 minutes of differentiation induction. Western blot was performed according to the method to confirm the change in the expression level of pp38 and p38.

The results are shown in FIG. 4A. In the figure pp38 refers to the amount of phosphorylated p38 protein, p38 refers to the amount of p38 protein of the base level. Actin refers to a control protein as a loading control. As shown in Figure 20 (S)-ginsenoside Rh2 can be seen to activate p38 in a time-dependent manner.

In addition, the cells prepared in the same manner as in 1-2. Were treated with 20 (S) -ginsenoside Rh2 (0, 10, 20, 40 uM) at the start of differentiation induction, and then 1-6. Western blot was performed according to the experimental method of to confirm the change in the expression level of pp38 and p38.

The results are shown in FIG. 4B. In the figure pp38 refers to the amount of phosphorylated p38 protein, p38 refers to the amount of p38 protein of the base level. Actin refers to a control protein as a loading control. As shown in Figure 20 (S)-ginsenoside Rh2 It can be seen that the activation of p38 in a concentration-dependent.

In addition, PKD activity inhibitors were applied to the cells prepared in the same manner as described in 1-2. After Go6976 was treated (10, 20uM) or untreated (-), Western blot was performed according to the experimental method of 1-6. At 60 minutes of differentiation induction to confirm the change in the expression level of pp38 and p38.

The results are shown in Figure 4C. In the figure pp38 refers to the amount of phosphorylated p38 protein, p38 refers to the amount of p38 protein of the base level. Actin refers to a control protein as a loading control. As shown in FIG. 4C, when both 20 (S) -ginsenoside Rh2 and Go6976 were not treated (-,-) or only Go6976 was treated (20 uM), p38 was not phosphorylated and was not activated. When only S) -ginsenoside Rh2 was treated, p38 was phosphorylated and activated, but when both 20 (S) -ginsenoside Rh2 and Go6976 were treated, the concentration of p38 was inhibited in Go6976.

In addition, SB294002, which is a p38 inhibitor, was applied to the cells prepared in the same manner as in 1-2, to the group treated with 20 (S) -ginsenoside Rh2 (40 uM) and the untreated group (-) at the onset of differentiation induction. After treatment (Calbiochem, CA, USA) (10, 20uM) or untreated (-), Western blot was performed at 60 minutes of differentiation induction according to the experimental method of 1-6. To confirm the change in the expression level of pp38 and p38. It was.

The results are shown in FIG. 4D. In the figure pp38 refers to the amount of phosphorylated p38 protein, p38 refers to the amount of p38 protein of the base level. Actin refers to a control protein as a loading control. As shown in the figure, when neither 20 (S) -ginsenoside Rh2 nor SB294002 was treated (-,-) or only SB294002 was treated (20 uM), p38 was not phosphorylated and was not activated, and 20 (S) When only ginsenoside Rh2 was treated, p38 was phosphorylated and activated, but 20 (S) -ginsenoside Rh2 and When all of SB294002 was treated, it can be seen that p38 activity was suppressed in a concentration-dependent manner to SB294002.

In addition, SB294002, which is a p38 inhibitor, was applied to the cells prepared in the same manner as described in 1-2. After treatment (10, 20uM) or untreated (-), ALP activity was measured according to the experimental method of 1-4. The results are shown in FIG. 4E. Fold increase refers to the relative ratio of 20 (S) -ginsenoside Rh2 and SB294002 untreated with ALP activity equal to 1.

In addition, SB294002, which is a p38 inhibitor, was applied to the cells prepared in the same manner as in 1-2, to the group treated with 20 (S) -ginsenoside Rh2 (40 uM) and the untreated group (-) at the onset of differentiation induction. After treatment (10, 20uM) or untreated (-), the degree of petrification was measured according to the experimental method of 1-5. After staining the degree of petrification through the Alizarin Red staining is destained and numerically measured by the method of measuring the absorbance is shown graphically in Figure 4E. Fold increase refers to the relative ratio of calcification of 1 with 20 (S) -ginsenoside Rh2 and SB294002 untreated.

As shown in FIG. 4E, it was confirmed that the ALP activity and mineralization increased by 20 (S) -ginsenoside Rh2 was suppressed concentration dependently in SB294002.

From these results, it was confirmed that 20 (S) -ginsenoside Rh2 promotes the activation of p38, a signaling substance involved in the mechanism of promoting bone formation, thereby promoting bone formation. As a result, osteoporosis, It can be seen that it is effective in fractures, bone formation disorders, malocclusion, and the like. In addition, fast and effective orthodontic treatment is possible by the bone formation promoting action of 20 (S) -ginsenoside Rh2 during orthodontic tooth movement. In other words, by promoting bone formation on the tension side during orthodontic tooth movement, bone formation by orthodontic force is possible in the alveolar bone and the entire periodontal tissue. In particular, the effect can be further increased by selectively applying 20 (S) -ginsenoside Rh2 on the tension side.

1-11. Effect of 20 (S) -ginsenoside Rh2 on AMPK and ACC (Acetyl-CoA carboxylase)

By confirming the effect of 20 (S) -ginsenoside Rh2 on the increase in the activity of AMPK, known as a cell differentiation promoting signal, experiments were carried out by the following method to confirm the effect of promoting the formation of osteoblasts by differentiation into osteoblasts of osteoblasts. It was. The increase of AMPK activity was confirmed by confirming the phosphorylation of AMPK and the degree of phosphorylation of ACC, a signaling material below.

The cells prepared in the same manner as in 1-2 were treated with 20 (S) -ginsenoside Rh2 (40 uM) at the onset of differentiation induction, followed by experiments of 1-6. At 0, 15, 30 and 60 minutes of differentiation induction. Western blot was performed according to the method to confirm the change in the expression level of pAMPK, AMPK, pACC, and ACC.

The results are shown in FIG. 5A. In the figure, pAMPK means the amount of phosphorylated AMPK protein, AMPK means the amount of AMPK protein at base level, pACC means the amount of phosphorylated ACC protein, and ACC means the amount of ACC protein at base level. Actin refers to a control protein as a loading control. As shown in Figure 20 (S)-ginsenoside Rh2 it can be seen that the time-dependent activation of AMPK and ACC.

In addition, the cells prepared in the same manner as in 1-2. Were treated with 20 (S) -ginsenoside Rh2 (0, 10, 20, 40 uM) at the start of differentiation induction, and then 1-6. Western blot was performed according to the experimental method of to determine the expression changes of pAMPK, AMPK, pACC, and ACC.

The results are shown in FIG. 5B. In the figure, pAMPK means the amount of phosphorylated AMPK protein, AMPK means the amount of AMPK protein at base level, pACC means the amount of phosphorylated ACC protein, and ACC means the amount of ACC protein at base level. Actin refers to a control protein as a loading control. As shown in Figure 20 (S)-ginsenoside Rh2 it can be seen that the activation of AMPK and ACC in a concentration-dependent manner.

In addition, Ara, an AMPK inhibitor, was applied to the cells prepared in the same manner as in 1-2. (Ara-A; adenine 9-β-D-arabinofuranoside; Sigma-Alcrich Inc. Mo. USA) after treatment (0.05, 0.1 mM) or untreated (-), eruption induction at 60 minutes of 1-6. Western blot was performed in accordance with the experimental method to confirm the expression changes of pAMPK, AMPK, pACC, and ACC.

The results are shown in Figure 5C. In the figure, pAMPK means the amount of phosphorylated AMPK protein, AMPK means the amount of AMPK protein at base level, pACC means the amount of phosphorylated ACC protein, and ACC means the amount of ACC protein at base level. Actin refers to a control protein as a loading control. As shown in the figure, when 20 (S) -ginsenosides Rh2 and Ara. Were not treated with (-,-) or only Ara. Was treated, AMPK and ACC were not phosphorylated and were not activated. When only S) -ginsenoside Rh2 was treated, AMPK and ACC were phosphorylated and activated, but when both 20 (S) -ginsenoside Rh2 and Ara. Were treated, the concentration-dependent AMPK and ACC activity was inhibited in Ara. Able to know.

In addition, Ara, an AMPK inhibitor, was applied to the cells prepared in the same manner as in 1-2. After treatment (0.05, 0.1 mM) or untreated (-), ALP activity was measured according to the experimental method of 1-4. The results are shown in FIG. 5 (D). Fold increase refers to the relative ratio of 20 (S) -ginsenoside Rh2 and Ara to 1 when Ara was not treated.

In addition, Ara, an AMPK inhibitor, was applied to the cells prepared in the same manner as in 1-2, to the group treated with 20 (S) -ginsenoside Rh2 (40 uM) and the untreated group (-) at the onset of differentiation induction. After treatment (0.05, 0.1 mM) or untreated (-), the degree of calcification was measured according to the experimental method of 1-5. After staining the degree of petrification through Alizarin Red staining is destained and numerically measured by the method of measuring the absorbance is shown in Figure 5 (D) as a graph. Fold increase means the relative proportion of 20 (S) -ginsenosides Rh2 with calcification of Ara as 1.

As shown in FIG. 5D, it was confirmed that the ALP activity and mineralization increased by 20 (S) -ginsenoside Rh2 was inhibited concentration-dependently in Ara.

In addition, PKD activity inhibitors were applied to the cells prepared in the same manner as described in 1-2, to the group treated with 20 (S) -ginsenoside Rh2 (40 uM) and the untreated group (-) at the start of differentiation induction. After Go6976 treatment (10, 20uM) or untreated (-), Western blot was performed at 60 minutes of differentiation induction according to the experimental method of 1-6. To confirm the expression changes of pAMPK, AMPK, pACC, and ACC. .

The results are shown in FIG. 5E. In the figure, pAMPK means the amount of phosphorylated AMPK protein, AMPK means the amount of AMPK protein at base level, pACC means the amount of phosphorylated ACC protein, and ACC means the amount of ACC protein at base level. Actin refers to a control protein as a loading control. As shown in the figure, when both 20 (S) -ginsenoside Rh2 and Go6976 were treated, it can be seen that AMPK and ACC activities are inhibited in a concentration-dependent manner to Go6976.

From these results, it was confirmed that 20 (S) -ginsenoside Rh2 promotes the activation of AMPK, which is known as a cell differentiation promoting signal, and promotes bone formation. As a result, osteoporosis, fracture, bone It can be seen that it is effective in formation disorders, malocclusion, and the like. In addition, fast and effective orthodontic treatment is possible by the bone formation promoting action of 20 (S) -ginsenoside Rh2 during orthodontic tooth movement. In other words, by promoting bone formation on the tension side during orthodontic tooth movement, bone formation by orthodontic force is possible in the alveolar bone and the entire periodontal tissue. In particular, the effect can be further increased by selectively applying 20 (S) -ginsenoside Rh2 on the tension side.

Preparation Example 1 Preparation of Pharmaceutical Composition

20 (S) -ginsenoside Rh2 of Example 1-1, 100 mg of corn starch, 100 mg of lactose, 2 mg of magnesium stearate were filled into gelatin capsules to prepare a capsule.

Preparation Example 2 Preparation of Food Composition

20 (S) -ginsenoside Rh2 (4 wt%), liquid fructose (0.5 wt%), oligosaccharide (2 wt%), sugar (2 wt%), and salt (0.5 wt%) of Example 1-1 After adding water to the remaining amount to adjust the homogeneous homogeneous sterilization to prepare a healthy drink.

Attach an electronic file to a sequence list

Claims (10)

delete delete A pharmaceutical composition for promoting tooth movement by promoting bone formation during orthodontic tooth movement containing 20 (S) -ginsenoside Rh2 as an active ingredient. delete delete Pharmaceutical composition for the treatment of traumatic fracture containing 20 (S) -ginsenoside Rh2 as an active ingredient. The composition of claim 6, wherein the treatment is by promoting bone formation. delete A food composition for promoting tooth movement by promoting bone formation during orthodontic tooth movement containing 20 (S) -ginsenoside Rh2 as an active ingredient. A food composition for improving traumatic fracture, comprising 20 (S) -ginsenoside Rh2 as an active ingredient.

Images