KR101306408B1 - A composition for promoting bone formation, comprising ginsenoside Rd as an active ingredient - Google Patents

Abstract

The present invention provides a composition for promoting bone formation containing ginsenoside Rd as an active ingredient.
The pharmaceutical or food composition according to the present invention promotes the expression of osteogenic genes and the secretion of osteogenic proteins, thereby promoting bone formation, bone diseases such as osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, It exhibits the effect of preventing, ameliorating or treating any disease treatable by bone defect, osteopenia, osteomalacia and resulting fractures, bone formation disorder, degenerative bone disease, malocclusion and bone formation or bone regeneration.

Description

A composition for promoting bone formation, comprising ginsenoside Rd as an active ingredient}

The present invention relates to a composition for promoting bone formation.

The bone of the human body maintains its homeostasis through the process of bone modeling and bone remodeling under the action of osteoblasts and osteoclasts. However, as the age increases, bone absorption increases due to various causes, resulting in vulnerable bone tissue, which causes various bone diseases such as osteoporosis and fractures. Therefore, there has been a need for the development of bone formation promoters for maintaining 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 mass and destruction of the bone matrix. The osteoporosis causes bone mass to decrease due to various causes and the risk of fracture increases continuously due to the deterioration of the microstructure of the bone tissue. do. Osteoporosis drugs used to date can be classified into bone resorption inhibitors and bone formation promoters.

Bone resorption inhibitors basically reduce fractures by preventing excess bone resorption by osteoclasts and increase bone mineral density, and bone formation promoters increase bone density and reduce fractures by increasing the number and differentiation of osteoblasts leading to bone formation. .

Commonly used osteoporosis therapies are a class of bone resorption inhibitors that weaken osteoclast function and induce death, and are mainly bisphosphonate series such as Fosamax (component name: alendronate) and actonel (component name: risedronate). It is used as a therapeutic agent. Parathyroid hormone is also used as an osteogenic agent. However, it is forbidden to use it for more than 18 months because the incidence of osteosarcoma increases with prolonged use.

Fracture is a state in which the continuity of bone, bone plate, or joint surface is abnormally broken, and is known to occur due to external shock such as trauma or metabolic abnormalities such as osteoporosis and bone cancer. In general, the fracture itself may not be a big problem, but it is known that secondary injury due to fracture, that is, complications.

Osteodystrophy, also known as Renal Osteodystrophy, is mainly caused by kidney disease. Nephrotic dysplasia is not a single disease but a group of diseases (hypothyroidism, osteomalacia, bone disease caused by aluminum, aplastic nephrotic dystrophy), and is known to be caused by one or several combinations. .

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 hepatocytes appear 12 hours later on the compression side during tooth movement and exist for 7 days. Acid phosphatase (ACP) and tartrate -Tartrate-resistant acid phosphatase (TRAP) activity is high, and the cells involved in bone formation show high alkaline phosphatase (ALP) activity and bone formation by orthodontic up to 21 days It is known to last. 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.

The present invention is to provide a pharmaceutical composition for promoting bone formation. In addition, the present invention is to provide a food composition for promoting bone formation.

The present inventors searched for various active substances for the treatment of bone-related diseases in natural products with excellent safety, and surprisingly, ginsenoside Rd, one of ginseng's active ingredients, expressed and secreted BMP-2, a bone formation promoting substance. It was confirmed to exhibit the effect of promoting bone formation to complete the present invention.

The present invention provides a pharmaceutical composition for promoting bone formation comprising ginsenoside Rd as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating bone diseases comprising ginsenoside Rd as an active ingredient.

The present invention also provides a food composition for promoting bone formation comprising ginsenoside Rd as an active ingredient.

The present invention also provides a food composition for the prevention or improvement of bone diseases comprising ginsenoside Rd as an active ingredient.

In the present specification, bone diseases include osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, bone defects, osteoplastic insufficiency, osteomalacia and resulting fractures, osteogenic disorders, degenerative bone diseases, and malocclusions, etc. All diseases treatable by formation or bone regeneration are included.

Ginsenoside Rd, an active ingredient in the composition of the present invention, is a kind of protopanaxdiol / PD / PPD ginsenoside, and a chemical formula is 2-O-beta-D-glucopyranosyl- ( 3beta, 12beta) -20- (beta-D-glucopyranosiloxy) -12-hydroxydammara-24-en-3-yl-beta-D-glucopyranoside, with the chemical structure same.

Ginsenoside Rd is known to have an adrenal cortex hormone secretion promoting effect, improves liver function (Korean Patent Publication No. 10-1994-0011007), and preventive effect of neurodegenerative diseases (Korean Patent Publication No. 10-2007-0033977) ), But no effect on bone formation has been revealed.

In the present invention, ginsenoside Rd may be separated from ginseng or red ginseng by a commonly used method known in the art, for example, from ginseng or red ginseng to be separated by thin layer chromatography, high performance liquid chromatography, and the like. May be, but is not limited thereto.

The ginseng may be a ginseng commonly used, preferably ginseng (Panax ginseng CA Meyer), Panax quinquefolium, Panax notoginseng, Panax japonicum, Panax trifolium or Himalayan It may be Panax pseudoginseng. More preferably, it may be ginseng, and may be ginseng or white ginseng, depending on the processing form. In addition, the red ginseng may be a commonly used red ginseng, for example, a high grade ginseng (earth g ginseng) and / or good grade ginseng (양) such as may be used. .

The content of ginsenoside Rd in the pharmaceutical composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the extent of progression, the condition of the patient, and the like, for example, 40 to 80% by weight, preferably 50 to 50% by weight of the total composition. 80 wt%, but is not limited thereto.

Ginsenoside Rd contained as an active ingredient in the pharmaceutical composition of the present invention may be formulated with a pharmaceutically or food-acceptable carrier, excipient or diluent, etc., and the formulation is described in Remington's Pharmaceutical Science. , Mack Publishing Company, Easton PA, et al.

As an embodiment of the present invention, the pharmaceutical composition containing ginsenoside Rd according to the present invention is oral dosage form of powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol and the like according to a conventional method respectively. It may be used in the form of external preparations, suppositories, or sterile injectable solutions.

Carriers, excipients and diluents which may be included here include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like, but are not limited thereto.

In addition, the pharmaceutical compositions of the present invention may include diluents or excipients such as fillers, extenders, binders, humectants, disintegrating agents, surfactants, and other pharmaceutically acceptable additives which are commonly used.

When formulated as a solid preparation for oral administration, tablets, pills, powders, granules, capsules, and the like are possible, and such solid preparations may include at least one excipient such as starch, calcium carbonate, It can be prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used.

When formulated as a liquid preparation for oral administration, suspensions, solvents, emulsions, syrups, and the like are possible, and various excipients, for example, wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin Etc. may be included.

When formulated into preparations for parenteral administration, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories are included. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like may be used, but are not limited thereto. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used, but are not limited thereto.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For the desired effect, the dosage of the composition of the present invention is 1 mg / kg to 100 mg / kg per day, preferably 1 mg / kg to 50 mg / day, based on adult ginsenoside Rd content. kg.

The administration may be carried out once a day or divided into several doses. However, the scope of the present invention is not limited by the dose and frequency of administration.

The pharmaceutical compositions of the present invention can be used in mammals such as rats, mice, livestock, humans, and the like, for example, orally, or intraperitoneally, rectally, intravenously, intramuscularly, subcutaneously, intrauterine dural or brain. It may be administered by injection or the like intravascularly (Intracerebroventricular).

The pharmaceutical composition of the present invention is selected from bone diseases, such as osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, bone defects, osteoplastic insufficiency, osteomalacia and resulting fractures, osteogenic disorders, degenerative bone diseases and malocclusion It can be used for the prevention or treatment of one or more diseases.

Accordingly, the present invention provides the use of ginsenoside Rd for the prophylaxis or treatment of the above-listed diseases, the use of ginsenoside Rd for the preparation of a medicament for the prevention or treatment of the above-listed diseases, a medicament for mammals including humans. Provided are methods for the prophylaxis or treatment of the diseases listed above comprising administering a pharmaceutically effective amount of ginsenoside Rd.

The present invention also provides a food composition for promoting bone formation, containing ginsenoside Rd as an active ingredient.

The food composition of the present invention can be used as a dietary supplement. "Health functional food" means a food manufactured and processed using raw materials or ingredients with functional properties useful to humans under No. 6727 of the Health Functional Food Act. "Functional" means the structure and It means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action with respect to function.

The food composition of the present invention may include a conventional food additive, and the suitability as a "food additive" is applied to the item in accordance with the General Regulations and General Test Methods of the Food Additives approved by the Food and Drug Administration, unless otherwise specified. Determined by the relevant standards and standards.

Examples of the items described in the "Food Additive Reduction" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid, natural additives such as color pigments, licorice extract, crystalline cellulose, high color pigments and guar gum, and L. And mixed preparations such as sodium glutamate preparation, noodle addition, preservative preparation and tar coloring preparation.

The health functional food composition of the present invention promotes bone formation or bone diseases, such as osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, bone defects, osteopenic insufficiency, osteomalacia and resulting fractures, bone formation disorders, degenerative bone diseases And it can be used in various ways, such as food and drink for the prevention or improvement of at least one selected from malocclusion, for example, can be used in various foods, beverages, gum, tea, vitamin complexes, health supplements, food additives, etc. It can be used in the form of a powder, granule, tablet, capsule or beverage. In addition to the above, it may be in any food form.

The food composition of the present invention promotes bone formation or bone diseases, such as osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, bone defects, osteogenic insufficiency, osteomalacia and the resulting fractures, osteogenic disorders, degenerative bone diseases and negative For the purpose of preventing or improving one or more selected during occlusion, the ginsenoside Rd may be included in an amount of about 40 to 80% by weight, preferably 50 to 80% by weight relative to the total weight of the composition.

In addition, the food composition of the present invention promotes bone formation or bone diseases, such as osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, bone defects, osteoplastic insufficiency, osteomalacia and resulting fractures, bone formation disorders, degenerative bone diseases And it can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, and the like for the purpose of prevention and / or improvement of one or more selected from malocclusion.

For example, the nutraceutical in the form of tablets is a mixture of ginsenoside Rd, excipients, binders, disintegrants, and other additives in a conventional manner and then compression molding with a lubricant or the like, or The mixture can be compression molded directly. In addition, the health functional food in the form of tablets may contain a mating agent and the like, if necessary, may be coated with a suitable coating agent.

Hard capsules of the health functional food in the form of capsules may be prepared by filling a conventional hard capsule with a mixture of ginsenoside Rd and an additive such as an excipient, or granules or peeled granules thereof. A mixture with side Rd and additives such as excipients can be prepared by filling a capsule base such as gelatin. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.

The dietary supplement in cyclic form may be prepared by molding a mixture of ginsenoside Rd, excipients, binders, disintegrants, etc. in a suitable manner, and may be coated with sucrose or other suitable coating agent, or starch, talc or suitable You can also be blessed with a substance.

The health functional food in the form of granules may be prepared by granulating a mixture of ginsenoside Rd, an excipient, a binder, a disintegrant, and the like in an appropriate manner, and may contain a flavoring agent, a copper, and the like as necessary. For the health functional food in the form of granules, No. 12 (1680 μm), No. 14 (1410 μm) and No. 45 (350 μm) were used for the next particle size test. Less than 5.0% and passing through the 45 can be less than 15.0% of the total amount.

The term definitions of the excipients, binders, disintegrants, glidants, copulation agents, flavoring agents, etc. are described in the literature known in the art and include those having the same or similar functions (Korean Pharmacopoeia, Munseongsa, Korea Pharmacy University Council, 5th edition, p33-48, 1989).

Ginsenoside Rd of this invention shows the outstanding bone formation promoting effect. Accordingly, pharmaceutical or food compositions containing ginsenoside Rd of the present invention may promote bone formation and / or bone diseases such as osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, bone defects, osteopenia, osteomalacia And thereby prevent, ameliorate, or treat a fracture, osteogenic disorder, degenerative bone disease, malocclusion, and any disease treatable by bone formation or bone regeneration.

1 is a graph confirming the increase in ALP activity in osteoblasts by ginsenoside Rd.
Figure 2 is a photograph confirming the increase in petrification in osteoblasts by ginsenoside Rd.
Figure 3 is a graph confirming the increase in concentration-dependent petrification in osteoblasts by ginsenoside Rd.
Figure 4 is a diagram confirming the effect of increasing the expression of osteogenic differentiation-related genes in osteoblasts by ginsenoside Rd.
5 is a graph confirming the increase in BMP-2 gene expression in osteoblasts by ginsenoside Rd.
Figure 6 is a graph confirming the increase in BMP-2 protein secretion in osteoblasts by ginsenoside Rd.
7 is a diagram confirming the effect of increasing phosphorylation of p38 MAPK by ginsenoside Rd.
8 is a diagram confirming the effect of increasing phosphorylation of AMPK and ACC by ginsenoside Rd.

Hereinafter, the present invention will be described in more detail through examples and test examples. However, these Examples and Test Examples are for illustrating the present invention, and the scope of the present invention is not limited to these Examples and Test Examples.

[ Manufacturing example ]

Ginsenoside Rd was purchased from Embo Research Institute (1511 Hongin Tower 1511, Bongmyeong-dong, Yuseong-gu, Daejeon, Korea) and dissolved in 0.1% DMSO (dimethylsulfoxide).

[ Example ]

Example  1. In mouse osteoblasts Gin Senocide Rd of Osteogenesis  Confirmation of promotion effect

(1) Induction of Cell Culture and Differentiation

Mouse osteoblast line MC3T3-E1 was purchased from Riken Cell Line Bank and added Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum and 1% penicillin / streptomycin (Invitrogen, CA, USA). The medium (Gibco BRL, USA) was passaged every 3 days at 37 degrees Celsius, 5% CO ₂ incubator. 6-well plates were then inoculated with ⁵ × 10 ⁵ cell numbers, respectively, followed by the addition of 50 ug / ml ascorbic acid (Sigma-Aldrich, MO, USA) and 10 mM beta-glycerophosphate (Sigma-Aldrich, MO, USA). Differentiation was induced for 24 days in DMEM medium.

Osteoblasts of 2.5 × 10 ⁴ cells / cm ² were inoculated into 6-well plates, and then two days later, differentiation-inducing media were added to induce differentiation. Differentiation-inducing control medium (Comparative) is DMEM medium with 50ug / ml ascorbic acid (Sigma-Aldrich, MO, USA) and 10 mM beta-glycerophosphate (Sigma-Aldrich, MO, USA) Rd treated group medium (experimental group) was used by adding 10, 20, 40 mM ginsenoside Rd thereto. Differentiation induction was carried out for a total of 24 days and the medium was changed every three days. The sample thus obtained was used for the following experiment.

(2) Gin Senocide Rd On by Osteogenesis  childhood Osteogenesis  Promotion effect confirmation- ALP  (alkaline 포스화제 Activity measurement experiment

ALP is known to be an indicator of bone formation in the early stages of bone formation.

The samples were taken on days 7, 14, 18 and 24, washed twice with cold PBS buffer, suspended in 0.1% Triton X-100 and lysed by an ultrasonic machine (Branson, USA). I was. The lysed cells were centrifuged at 4 degrees Celsius and 12000 rpm for 20 minutes, and the supernatant was recovered and quantified by Bradford method. The same amount of protein was prepared using the ALP kit (Sigma-Aldrich, MO, USA) according to the manufacturer's instructions. ALP activity was measured at 405 nm. The results are shown in FIG.

Figure 1A is the result of measuring the time-dependent ALP activity to confirm the bone formation promoting effect by ginsenoside Rd, Figure 1B is a measure of the concentration of ALP activity by the concentration ginsenoside Rd on day 14 of differentiation. As confirmed in FIG. 1, ginsenoside Rd exhibited the highest ALP activity on day 14 of differentiation and was also found to be concentration dependent.

(3) Gin Senocide Rd On by Osteogenesis  Identification of stimulating effects-petrification ( mineralization  Measurement experiment

Calcium accumulated extracellularly during bone formation was confirmed by staining with Alizarin Red S (Sigma-Aldrich, USA) and VonKossa (Invitrogen, CA, USA). The cells differentiated for 24 days were fixed with 10% formalin for 1 hour, and then stained for 10 minutes with 1% alizarin red solution and 1% Von Kossa solution, respectively, for the two samples. The results are shown in Figs. 2 and 3.

As can be seen in Figures 2 and 3, ginsenoside Rd was confirmed to increase the calcification effect in a concentration-dependent manner.

(4) Gin Senocide Rd On by Bone differentiation  Confirmation of Increased Promoting Gene Expression mRNA  Separation and RT - PCR

To identify gene changes during the differentiation process, total RNA was isolated from the stages of differentiation (cells on days 0, 7, 14 and 24) using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. BMP-2 is an early stage of bone formation, and to confirm the BMP-2 gene change, the same experiment was performed by obtaining total RNA from cells on day 5 of differentiation. The cultured cells were washed twice with cold PBS and then lysed with 1 ml of Trizol reagent. 200 μl chloroform was added and mixed, followed by centrifugation at 4 ° C. and 12000 rpm for 20 minutes to separate the supernatant. The same amount of isopropanol was added to the separated supernatant, followed by centrifugation at 4 ° C. and 12000 rpm. The supernatant was discarded and the remaining pellet was washed three times with 70% ethanol and RNA was isolated. For RT-PCR, the same amount of total RNA (5 μl) was added to the RT-PCR amplification kit and reacted for 60 minutes at 45 degrees Celsius to prepare cDNA. cDNA was amplified by a real-time PCR method using the primers of Table 1 below. Real-time PCR was performed using SYBR Green reagent (Roche, Basel, Switzerland) on the cDNA according to the manufacturer's instructions. Specifically, 25 μl of SYBR green and 1 μl of each primer were added to the same amount of cDNA, and then amplified by reacting for 30 seconds at 5 degrees and 1 hour at 50 degrees Celsius.

[Table 1] Bone Formation Related Gene Primers

* HPRT: Control gene in quantitative analysis

RT-PCR results, bone differentiation promoting gene (ALP, OCN, Col-I) expression results are shown in Figure 4. In addition, BMP-2 gene expression results are shown in FIG.

As shown in FIG. 4, mRNA expression of ALP, an early indicator of bone differentiation, was highest at 14 days of differentiation (FIG. 4A), which was found to be concentration dependent on ginsenoside Rd (FIG. 4D). On the other hand, mRNA expressions of OCN and Col-1, which are indicators of mid and late bone differentiation, began to increase on the 14th day of differentiation and showed the highest expression on the 24th day of differentiation (Figs. 4B and 4C), which were also concentration-dependent on ginsenoside Rd. Confirmed (FIGS. 4E, 4F).

In addition, as shown in Figure 5, it was confirmed that the mRNA expression of BMP-2 known as the differentiation promoting agent increases the mRNA expression of BMP-2 known as the differentiation promoting agent concentration-dependently to ginsenoside Rd.

(5) Gin Senocide Rd In osteoblasts by BMP -2 Increase secretion effect

BMP-2 is known as an indicator of bone formation in the early stages of bone formation.

The supernatant was recovered by spinning off the medium on day 5 of the differentiation. Measurement of secreted BMP-2 in the medium was measured by ELISA using the BMP-2 measurement kit (R & D Systems, Cat. No. DBP200, USA) according to the manufacturer's instructions. The results are shown in Fig.

As can be seen in Figure 6, ginsenoside Rd was found to promote the secretion of BMP-2 known as the differentiation promoting agent in a concentration-dependent manner.

As confirmed through the above example, it was confirmed that the bone differentiation promoting effect of ginsenoside Rd. Further experiments were performed as follows to elucidate the mechanism of promoting bone differentiation by ginsenoside Rd.

Example  2. Mouse In osteoblasts Gin Senocide Rd of Osteogenesis  Confirmation of related mechanism

(One) Gin Senocide Rd On by p38 MAPK  Check for active changes

Two days after inoculation of 2.5 x 10 ⁴ cell / cm ² osteoblasts into 6-well plates, Ginsenoside Rd was treated with concentrations (0, 10, 20, and 40 mM), and 0.5h, 1h, 3h hourly samples. Was collected and Western blotting was performed. For protein analysis, the samples were homogenized with Lysis buffer and the protein was quantified using Bio Rad assay reagent (Bio-Rad, USA). After the same amount of protein (50 mg) was separated by 8% SDS-PAGE, the gel was transferred to the membrane (Millipore, Cat. No L IPVH00010) and blocked for 2 hours at room temperature with 5% scheme milk. It was then reacted overnight at 4 degrees Celsius in a solution of phospho-p38MAPK antibody (cell signaling, 9212) diluted 1: 1000 ratio. After washing three times with Tris-buffered saline Tween 20 (TBST) and reacting with a secondary antibody (Santa Cruz, sc-2313) diluted at a ratio of 2000 for 2 hours at room temperature, it was washed three times with TBST. Thereafter, the resultant was printed on an X-ray film using an ECL solution (Amersham, Sweden) to confirm whether p38 MAPK was activated. The results are shown in Fig.

As shown in Figures 7A and 7B, ginsenoside Rd was found to enhance the phosphorylation of p38 MAPK in a time- and dose-dependent manner. In addition, the phosphorylation of p38MAPK was significantly reduced after 2 hours of pretreatment with 0.5 mM p38 MAPK inhibitor SB203080 (Invitrogen, Cat.No. PHZ1253, USA) (FIG. 7C), and SB294002 was also ginsenoside Rd It was confirmed that significantly reduced the increased ALP activity and calcification by (Fig. 7D; measured in the same experiment as Example 1- (2), (3)). This indicates that ginsenoside Rd promotes osteogenic differentiation through the p38 MAPK activation mechanism.

(2) Gin Senocide Rd On by AMPK  Check for active changes

Two days after inoculation of 2.5 x 10 ⁴ cell / cm ² osteoblasts into 6-well plates, Ginsenoside Rd was treated with concentrations (0, 10, 20, 40 mM) at 0.5 h, 1 h, and 3 h hourly samples. Was collected and Western blotting was performed. Western blotting procedures were as described above except the activity of AMPK was confirmed using phospho-AMPK antibodies (cell signaling, Cat. No. 2535, USA).

As can be seen in Figures 8A and 8B, ginsenoside Rd was found to enhance the phosphorylation of AMPK in a time- and dose-dependent manner. In addition, when AMPK phosphorylation was inhibited by pretreatment with Compound C, 20 μM, which is an AMPK inhibitor (FIG. 8C), it was confirmed that the increased ALP activity and calcification by Ginsenoside Rd were significantly reduced (FIG. 8D). This indicates that ginsenoside Rd promotes osteogenic differentiation through AMPK activation mechanism.

Attach an electronic file to a sequence list

Claims (8)

Ginsenoside Rd containing as an active ingredient, a pharmaceutical composition for promoting bone formation. A pharmaceutical composition for preventing or treating bone diseases, comprising ginsenoside Rd as an active ingredient. The pharmaceutical composition according to claim 2, wherein the bone disease is selected from osteoporosis, osteoporotic fracture, diabetic fracture, nonunion fracture, bone defect, osteoplasia, osteomalacia and the resulting fracture, bone formation disorder, degenerative bone disease, or malocclusion Composition. The pharmaceutical composition according to any one of claims 1 to 3, which contains 40% to 80% by weight of ginsenoside Rd based on the total weight of the composition. Food composition for promoting bone formation, containing ginsenoside Rd as an active ingredient. Food composition for the prevention or improvement of bone diseases, containing ginsenoside Rd as an active ingredient. 7. The food product of claim 6, wherein the bone disease is selected from osteoporosis, osteoporotic fracture, diabetic fracture, nonunion fracture, bone defect, osteopenia, osteomalacia and the resulting fracture, bone formation disorder, degenerative bone disease, or malocclusion Composition. The food composition according to any one of claims 5 to 7, which contains 50 to 80% by weight of ginsenoside Rd based on the total weight of the composition.

Images