KR20170107791A - Composition for promoting osteogenesis comprising tectorigenin - Google Patents

Abstract

The present invention relates to a composition for promoting osteogenesis and, more specifically, to a composition for promoting osteogenesis and comprising a compound isolated from a Belamcandachinensis extract as an active ingredient. The compound isolated from a Belamcandachinensis extract is a compound of chemical formula 1. The composition for promoting osteogenesis promotes differentiation of osteoblasts, and suppresses differentiation of osteoclasts.

Description

TECHNICAL FIELD The present invention relates to a composition for accelerating bone formation comprising tectorigenin,

TECHNICAL FIELD The present invention relates to a composition for promoting bone formation, and more particularly to a composition for promoting bone formation comprising tectorigenin isolated from a naturally occurring extract.

Bones are an active organization that constantly changes throughout their lives. The bones are divided into the external cortical bone (fine bone) and the internal shoju bone (cancellous bone, sponge bone) with the naked eye. The cortical bone has a strong physical strength to protect and support the body. To maintain a constant change of.

Remodeling of bones is a phenomenon in which old bone is destroyed and removed (bone resorption), and fixation (osteogenesis) that replaces new bone is repeated throughout life, even after bone growth has ceased. The balance between osteoblast and osteoclast balances the bone resorption and osteogenesis to maintain bone homeostasis and the calcium concentration in the blood is kept constant. When calcium is lacking in the blood, The absorption is increased to release the calcium of the bone into the blood. If the bone resorption continues, the bone is weakened and the disease such as osteoporosis occurs.

Particularly, osteoporosis is caused by postmenopausal osteoporosis, which is caused by an increase in bone resorption due to activation of osteoclast due to rapid hormone change due to menopause, and decreased osteoblast function due to aging. It can be classified as geriatric osteoporosis. Since the fracture caused by osteoporosis limits physical activity and the hip fracture may lead to death, diagnosis and treatment before osteoporotic fracture are important.

In the United States, about 30 percent of women in postmenopausal women have osteoporosis, about 50 percent have osteopenia, and about 26 million people have osteoporosis-related fractures, the World Health Organization (WHO) reports. In the United States, these osteoporosis patients are expected to increase by about 2% each year. In Korea where rapid aging is taking place, osteoporosis patients are increasing rapidly.

Osteoporosis treatments are classified as antiresorptive drugs and bone formation promoters. Bone resorption inhibitors include bisphosphonates, selective estrogen receptor modulators (SERMs), calcitonin, and the like, and parathyroid hormone (PTH) and fluoride agents are examples of therapeutic agents for controlling osteoclast formation and activity.

Bisphosphonate, which is the biggest market in the osteoporosis treatment market, has a disadvantage in that it needs to be taken on an empty stomach and has side effects such as esophagitis and esophageal perforation. Selective estrogen receptor modulators include, but are not limited to, raloxifene, droloxifene, and lasofoxifene, which have side effects that increase the risk of breast and uterine cancer induction. Calcitonin is expensive and difficult to administer, and calcium preparations have few side effects but are limited to prevention rather than osteoporosis treatment.

The osteoporosis medicines developed so far have only a limited effect on osteogenesis except for the parathyroid hormone, so that they prevent the progress of the symptoms rather than the treatment effect. In addition, the parathyroid hormone agent having an osteogenesis effect promotes osteogenesis to obtain a therapeutic effect, but its safety is not established for long-term use and its use is limited. Therefore, for the fundamental treatment of osteoporosis, recovery of lost bone mass is indispensable, and development of a small molecule bone formation drug for osteoporosis treatment is urgent.

The present inventors have conducted research for development of a low molecular weight compound that promotes osteoblast differentiation that is essential for osteogenesis, fracture recovery, and tooth growth and development, and has succeeded in separating a novel compound derived from a natural plant, Formation effect.

It is an object of the present invention to provide a novel compound capable of promoting osteoblast differentiation essential for bone formation and inhibiting osteoclast differentiation and promoting bone formation in order to solve the problems of the above-described prior art.

It is another object of the present invention to provide a composition for preventing and treating bone diseases, which comprises the novel compound.

According to one aspect of the present invention, there is provided a composition for promoting bone formation comprising a compound of the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

In one embodiment, the compound of Formula 1 may be isolated from an extract of Belamcanda chinensis .

In one embodiment, the composition can promote osteoblast differentiation and inhibit osteoclast differentiation.

In one embodiment, the composition is capable of activating a BMP signaling pathway.

In one embodiment, the composition may increase one or more phosphorylation levels selected from the group consisting of Smad1, Smad5, and Smad8.

In one embodiment, the composition is capable of upregulating the expression of Runx2 and osterix.

In one embodiment, the composition can activate the BMP signaling pathway by increasing the phosphorylation level of ERK or JNK.

In one embodiment, the composition may inhibit the RANK signaling pathway.

In one embodiment, the composition can downregulate the expression of c-Fos or NFATc1.

In one embodiment, the composition may be for the prevention or treatment of bone disease.

In one embodiment, the bone disease may be selected from the group consisting of osteoporosis, osteogenesis imperfecta, osteomalacia, osteopenia, fracture, bone defect, and hip arthropathy.

According to the present invention, the composition for promoting osteogenesis, including a naturally occurring chemical, has low toxicity in the body and is excellent in osteogenesis promoting ability, and thus can be usefully used for prevention and treatment of bone diseases.

It should be understood that the effects of the present invention are not limited to the effects described above, but include all effects that can be deduced from the description of the invention or the composition of the invention set forth in the claims.

Figure 1 shows the structure of the isolated tectorigenin. (A) The chemical structure of tectorigenin. (B) HPLC chromatogram results of tectorigenin.
Fig. 2 shows the cytotoxicity of tectorigenin and the differentiation effects of primary cultured osteoblasts. (A) MTT assay to analyze cell viability. (B) ALP activity. (C) Alizarin red staining results. (D) marker gene.
FIG. 3 shows the cytotoxicity and cell differentiation effects of tectorigenin in human periodontal ligament cells. (A) MTT assay to analyze cell viability. (B) ALP activity. (C) Alizarin red staining results. (D) marker gene.
Figure 4 shows the effect of tectorigenin on BMP and MAPK signaling pathway activation. (A) Cells were cultured for 3 days in OM (osteogenic medium) with the indicated concentrations of tectorigenin. (B) cells were incubated in osteogenic medium for 60 min with the indicated concentrations of tectorigenin. (C) Cells were cultured in OM (osteogenic medium) for 30 min with the indicated concentrations of tectorigenin.
Figure 5 is an evaluation of the in vivo bone regeneration effect of tectorigenin. (A) Micro CT images of the skull defect after 6 weeks. (B) The degree of new bone formation was determined by 3D micro CT. (C) The histological image (left) and enlarged image (right) of the H & E staining skull defect are shown.
Figure 6 shows the effect of tectorigenin on RANKL mediated osteoclast differentiation in mouse BMM. (A) TRAP staining results. (B) The number of TRAP-positive polynuclear cells (MNC) containing three or more nuclei was quantified. (C) RT-PCR results for osteoclast-specific marker genes.
Figure 7 shows the effect of tectorigenin on RANKL mediated MAPK. (A) BMM was incubated with RANKL and 100 μM tectorigenin for 30 min. (B) BMM was incubated with RANKL and 100 μM tectorigenin for 6 hours.
Figure 8 assesses the effect of tectorigenin on bone resorption induced by LPS. (A) A micro CT image that visualizes and quantifies the absorption of the skull. (B) The relative bone resorption rate was determined by 3D micro CT (BV / TV).
Figure 9 is a schematic representation of the mechanism of action of tectorigenin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. When an element is referred to as "comprising ", it means that it can include other elements, not excluding other elements unless specifically stated otherwise.

Unless otherwise defined, can be performed by molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing and routine techniques commonly used in the art of recombinant DNA within the skill of those skilled in the art. These techniques are known to those skilled in the art and are described in many standardized textbooks and references.

Various scientific dictionaries, including the terms contained herein, are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing of the present application, some methods and materials have been described. It is not intended that the invention be limited to the particular methodology, protocols, and reagents, as they may be used in various ways in accordance with the context in which those skilled in the art use them.

Hereinafter, the present invention will be described in more detail.

An aspect of the present invention provides a composition for promoting bone formation comprising a compound of the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

In one embodiment, the compound of Formula 1 may be isolated from an extract of Belamcanda chinensis .

The salt may be an acid addition salt formed by a pharmaceutically acceptable free acid. The acid addition salt can be prepared by a conventional method, for example, by dissolving the compound in an excess amount of an aqueous acid solution and precipitating with a water-miscible organic solvent (for example, methanol, ethanol, acetone or acetonitrile). Further, the same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated, the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration.

The free acid may be an organic acid or an inorganic acid. For example, the inorganic acid may be hydrochloric acid, bromic acid, sulfuric acid, or phosphoric acid. The organic acid may be citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, wherein the organic acid is selected from the group consisting of acetic acid, acetic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4- toluenesulfonic acid, benzenesulfonic acid, salicylic acid, nicotinic acid, isonicotinic acid, Or aspartic acid, but are not limited thereto.

The compound of Formula 1 is a main compound of Belamcanda chinensis and is called tectorigenin and is a kind of isoflavone derivative. Biotechnol. Biochem., 65 (4), 939-942, 2001) and anti-inflammatory effects (Archer Pharm. Res., 25, 320-324, 2002) (Biol. Biophys. Acta, 1438, 399-407, 1999). Recent studies have also reported that tectorigenin binds to estrogen receptors and is capable of estrogen-like action (Biol. Pharm. Bull., 25, 48-52, 2002).

Despite the various physiological effects of the tectorigenin, it has not been found that the tectorigenin can directly act on the signal pathway involved in osteoblast and osteoclast differentiation to promote bone formation. The present inventors first discovered the mechanism of action of tectorigenin on bone formation, and provided a method for preventing and treating bone diseases based on the above understanding.

Refers to an extract obtained by extracting the alabaster with water or an organic solvent and can be extracted with water, an alcohol having 1 to 6 carbon atoms, a nucleic acid, chloroform, methyl acetate or butanol as a solvent.

In one embodiment, the snack extract may be obtained by using alcohol as a solvent. The alcohol may be an aliphatic alcohol having 1 to 6 carbon atoms, and may be methanol, ethanol, isopropanol, butanol, hexane or the like, but is not limited thereto.

The alcohol extract may be dispensed into an organic solvent and water. The organic solvent may be selected from aliphatic hydrocarbons having 1 to 10 carbon atoms, aliphatic halogenated hydrocarbons having 1 to 10 carbon atoms, and esters having 2 to 10 carbon atoms, preferably ethyl acetate.

The fractionated ethyl acetate fraction can then be separated via silica gel column chromatography. The silica gel column chromatography can use a mixed solution of n-hexane and dichloromethane as an eluent, and the volume ratio of the mixed solution can be sequentially adjusted from 20: 1 to 2: 1.

Further, the eluate obtained by the silica gel column chromatography can be subjected to silica gel column chromatography again using n-hexane and dichloromethane as an eluent (1: 2 to 1: 3 by volume) The eluted eluent can be used as eluent (3: 1 to 5: 1 by volume) of n-hexane and ethyl acetate to finally obtain the compound of formula (1).

On the other hand, the composition can promote osteoblast differentiation. The osteoblast is a cell for synthesizing and secreting a bone matrix and calcining the bone tissue by depositing an inorganic salt such as calcium or magnesium ion on the matrix. The osteoblast is a region where osteogenesis, bone growth or bone regeneration Found. The osteoblast cells bind to the bone tissue formed by the osteoblast during the osteogenesis process and become bone cells.

The composition may activate the BMP signaling pathway. In one embodiment, the composition can increase one or more phosphorylation levels selected from the group consisting of Smad1, Smad5, and Smad8 and upregulate the expression of Runx2 and osterix. In addition, the composition may activate the BMP signaling pathway by increasing the phosphorylation level of ERK or JNK.

The composition activates the signal pathway mediated by the BMP, thereby promoting osteoblast differentiation.

Bone Morphogenetic Protein (BMP) is a peptide growth factor belonging to the Transforming Growth Factor β (TGF-β) phase and promotes the differentiation of stem cells into osteocytes and chondrocytes in mammals (Jiwang Zhang, Linheng Li, BMP signaling and stem cell regulation (2005) Developmental Biology 284 1-11).

Receptor activation by BMP2 induces phosphorylation of SMAD and permits intracellular entry of pSMAD 1/5/8. The BMP may interact with RUNX2, which is important for osteocyte formation, remodeling, growth and development processes, to enhance the ability to regulate the downstream factors. ERK, p38 and JNK, which are activated by the BMP signaling pathway, are involved in the regulation of p300, which plays an important role in the stability and transcription factor function of RUNX2, and can promote osteoclast differentiation.

The osteoblasts may be affected by transcription factors such as Runx2 and osteotelia, which are known as key transcription factors in bone synthesis and formation, and the BMP signal pathway can be regulated by the action of various biomolecules. Runx2 overexpressed in mesenchymal stem cells can promote osteoblast differentiation.

RUNX2 knockout mice, in which the RUNX2 gene has been removed, may be delayed in the maturation process of osteoblasts and may stop ossification of intracellular and cartilage (Komori et al., 1997; Otto et al., 1997). RUNX2 can regulate the expression of various genes such as alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OP), and type I collagen, which are specific to osteoblasts. When mature osteoblasts are inhibited in the expression of RUNX2, their activity may be lowered and bone formation and bone density may be significantly reduced (Ducy et al., 1999).

The tectorigenin activates the BMP signal pathway directly involved in osteoblast differentiation and increases the activity of various proteins involved in the signal pathway, thereby promoting bone formation.

In addition, the composition can inhibit osteoclast differentiation. The osteoclast is a large polynuclear cell that destroys or absorbs bone tissue that is unnecessary in the process of bone growth of a vertebrate, and may lower bone density and cause various bone diseases including osteoporosis.

In one embodiment, the composition may inhibit the RANK signaling pathway and down regulate the expression of c-Fos or NFATc1.

Differentiation of the osteoclast can be initiated by binding RANKL with the RANK receptor. Specifically, if the RANKL binds to RANK, biological molecules such as TRAF6 (tumor necrosis factor receptor-associated factor 6) can be activated. The TRAF family members TRAF2, TRAF5, and TRAF6 can activate transcription factors such as NF-Kb and AP-1 (activator protein-1) required for osteoclast differentiation.

The TRAF6 activated by the RANKL signal is composed of MAPKs, NF-kB, c-Fos, Fra-1, CREB, including MAPKs including PI3K, transforming growth factor beta-activated kinase (TAK1), Akt / PKB and JNK, (cyclic-AMP-responsive-element-binding protein), NFATc1 (nuclear factor of activated T cells cytoplasmic 1). By this pathway, osteoclast-specific genes such as cathepsin K, tartrate-resistant acid phosphatase (TRAP), calcitonin receptor and osteoclast-associated receptor (OSCAR) can be expressed.

The tectorigenin inhibits the RANK signaling pathway directly involved in osteoclast differentiation and can promote osteogenesis by reducing or eliminating the activity of various signal molecules involved in the signal pathway.

In one embodiment, the composition may be for the prevention or treatment of bone disease, and the bone disease may be metabolic bone disease or orthopedic bone disease.

The above-mentioned " metabolic bone disease " means a condition or disease caused by excessive production or activity of osteoclasts, and may include bone loss diseases. The term "lowering bone disease" refers to a condition or disease in which a decrease in bone mass accompanying symptoms such as a decrease in bone density and softening of bone tissue occurs.

The bone disease may be selected from the group consisting of osteoporosis, osteoarthritis, osteomalacia, osteopenia, fracture, bone defect and hip joint hypoplasia, but is not limited thereto.

The composition can also be used for the prevention and treatment of periodontal disease. The term "periodontal disease" comprehensively refers to a disease affecting the alveolar bone surrounding tissues supporting or engaging the alveolar bone or the alveolar bone with the gingiva, and more specifically, a pathogenic metabolite Which may impair the structure of the tooth, damage the germ cell tissue, weaken the immunity of the gingival tissue, and proliferate the periodontal bacteria and cause the lesion to repeatedly expand.

The periodontal disease may be classified into gingivitis and periodontitis according to the region of the lesion. For example, the gingivitis refers to a disease in which the lesion develops in a soft tissue, and periodontitis may be a disease in which the lesion appears in a region extended to the periphery of the gum and the gum bone.

The composition may be used in one or more materials selected from the group consisting of artificial bone, artificial joint, bone fixation material, bone substitute, bone restoration material, bone filler and bone graft material.

The composition for promoting bone formation can be used for prevention and treatment of bone disease or periodontal disease and can be manufactured in a form that can be easily applied to artificial bone, bone fixation material, bone substitute, bone restoration material, bone filler or bone graft material .

The bone graft material or the bone substitute can be used to fill a space in the bone tissue in a narrow sense, and can be widely used to replace a part of a bone or a tooth. Therefore, the composition for promoting bone formation can be used in the form of putty, paste, moldable strip, block, chip or the like using a method such as compression, compression, pressure contact, packing, pressing, And may be formulated into gels, granules, pastes, tablets, pellets, etc., and may be used in powder form.

The compositions can be administered in the form of oral delivery, parenteral delivery. The compound of Formula 1 may be administered systemically or topically, and the administration may include oral administration and parenteral administration. When the compound is administered as a composition, it may be formulated with a suitable amount of a pharmaceutically acceptable vehicle or carrier to provide a suitable dosage form.

On the other hand, the composition may further comprise a carrier, an excipient, and a diluent used in the production of the pharmaceutical composition.

Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, But are not limited to, cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

In addition, the composition may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, external preparation, suppository and sterilized injection solution.

Solid formulations for oral administration may be tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, , Lactose, or gelatin. In addition to the above excipients, lubricants such as magnesium stearate and talc may be used.

The liquid preparation for oral administration may be a suspension, a solution, an emulsion, a syrup, etc. In addition to water and liquid paraffin which are simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be used.

The preparation for parenteral administration may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository. Examples of the non-aqueous solutions and suspensions may include injectable esters such as propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, and glycerogelatin.

The present invention will be further described with reference to the following examples, but it should be apparent that the present invention is not limited by the following examples.

Example: Isolation of tectorigenin

After 2.0 kg of dried rootstock rootlets were divided into three portions of 3 L of methanol seven times, the solvent was concentrated under reduced pressure to obtain 537.1 g of methanol extract.

The methanol extract was separated into a fraction of n-hexane (38.3 g), methylene chloride (94.1 g), ethyl acetate (27.8 g) and n-butanol (114.9 g) The separated methylene chloride fractions were subjected to silica gel column chromatography using CH ₂ Cl ₂ -CH ₃ OH solvent (CH ₂ Cl ₂ : CH ₃ OH = 7: 3 (v / v)) to separate tectorigenin.

The separated tectorigenin was obtained in the form of an amorphous light yellow powder after solvent removal and washing.

(-) - ESI-MS m / z 299 [MH] -, 1 H-NMR (DMSO- d 6, 500MHz) δ: 8.32 (1H, s, H-2), 7.37 (2H, d, J = 8.7 Hz, H-2 ', 6 '), 6.82 (2H, d, J = 8.7Hz, H-3 ', 5'), 6.49 (1H, s, H-8), 3.74 (3H, s, OCH 3 ); _{^{13 C-NMR (DMSO- d 6}} , 125MHz) δ 181.1 (C-4), 158.0 (C-9), 157.9 (C-4 ''), 154.6 (C-2), 153.8 (C-7), 153.3 (C-5), 131.9 (C-6), 130.7 (C-2 ', 6'), 122.3 , 105.4 (C-10), 94.4 (C-8), and 60.5 (OCH 3).

Experimental Example 1: Evaluation of cytotoxicity of tectorigenin

MTT assays were performed to assess the cytotoxicity of tectorigenin following over 14 days culture. Tectorigenin did not show cytotoxicity up to a concentration of 100 μM (FIG. 2A).

Alkaline phosphatase activity (ALP activity), mineralization, and mRNA expression of various markers were evaluated to examine the osteogenic properties of tectorigenin.

The initial (ALP activity) and terminal (calcium deposition) differentiation markers increased in proportion to the concentration when the primary cultured osteoblasts were treated with tectorigenin for 14 days (Fig. 2B, 2C).

Tectorigenin also up-regulates bone-specific transcription factors such as Runx2 and osterix as well as mRNA differentiation markers such as ALP, osteocalcin (OCN) and osteopontin (OPN) 2D).

The tectorigenin promoted osteoblast differentiation without periodic cytotoxicity in periodontal ligament cells (PDLC).

Experimental Example 2: Effect of tectorigenin on osteoblast differentiation

The present inventors analyzed whether the differentiation of osteoblasts induced by tectorigenin is an important factor in the cells involved in bone regeneration and analyzed the effect of tectorigenin on the osteoclast ligament cells. Contributed.

Tectorigenin increased ALP activity, calcified nodule formation, and mRNA levels of various markers, while not affecting cell growth (Figures 3A-D).

Experimental Example 3: Effect of tectorigenin on BMP and MAPK signal pathway activation

To elucidate the mechanisms involved in the osteoblast differentiation of tectorigenin, we examined the effects of tectorigenin on bone morphogenetic protein (BMP) and mitogen-activated protein kinase (MAPK) signaling pathways.

Administration of tectorigenin in primary cultured osteoblasts increased the levels of mRNA expression of Bmp2, Bmp4, and Smad4 (Fig. 4A).

In addition, in primary cultured osteoblasts, tectorigenin increased the phosphorylation level of Smad1 / 5/8, ERK and JNK downstream signaling molecules of the BMP signaling pathway (Fig. 4B, 4C).

Experimental Example 4: Effect of tectorigenin on bone regeneration

To confirm the effect of tectorigenin on bone regeneration in vivo , the inventors performed micro-CT (μCT) and histological analysis after treating cortical deficient mice with tectorigenin, BMP-2 or a control group .

According to micro-CT (μCT) analysis, tectorigenin and BMP-2 increased bone formation over a period of 6 weeks, while the control group induced minimal bone formation (FIG. 5A).

According to the concentration-based μCT analysis, tectorigenin significantly increased the new bone volume fraction compared to the control (P <0.05) but was found to be lower than that of rh-BMP2 (FIG. 5B).

According to the histological examination, osteogenesis of the defect site was confirmed by treatment with tectorigenin, unlike the control region which is mostly fibrous tissue (Fig. 5C).

In addition, the large bone mass between the skull defect in the BMP group included dense cancellous bone.

Experimental Example 5: Inhibitory effect of tectorigenin on osteoclastogenesis

In order to analyze the effect of tectorigenin on osteoclast differentiation derived from BMM (bone marrow micrometastasis) of mice, the present inventors used tectorigenin in the presence of M-CSF (30 ng / mL) and RANKL (100 ng / (0 to 100 [mu] M) for 5 days.

Tectorigenin inhibited RANKL-mediated osteoblast differentiation (Fig. 6A). In addition, the number of tartrate-resistant acid phosphatase (TRAP) -positive cells was significantly reduced by tectorigenin (Fig. 6B).

Cytotoxicity assays were performed to determine if the decrease in osteoclastogenesis by tectorigenin was due to the effect of survival of tectorigenin on progenitor cells.

Tectorigenin did not show cytotoxicity at the tested concentrations. In addition, tectorigenin inhibited osteoclastogenesis-related marker genes such as TRAP, cathepsin-K, and MMP-9 (FIG. 6C).

Experimental Example 6: Inhibitory effect of tectorigenin on RANK signal pathway

The inventors investigated the MAPK and transcription factors involved in osteoclast differentiation in order to elucidate the mechanism of osteoclastogenesis inhibition of tectorigenin.

Tectorigenin inhibited RANKL-induced phosphorylation of p38 and ERK in BMM of mice, but did not affect JNK (Fig. 7A). Tectorigenin completely inhibited RANKL-induced NFATc1 and c-Fos proteins (Fig. 7B).

Experimental Example 7: Inhibitory effect of tectorigenin on bone resorption

The present inventors have investigated the in vivo effect of tectorigenin using a bone erosion animal model. The skull of mice was subcutaneously injected with lipopolysaccharide (LPS) along with tectorigenin or a control group.

According to micro CT (μCT) analysis, bone loss induced by LPS was clearly restored in the skull of mice that received tectorigenin together (FIG. 8A).

The microstructure index (BV / TV) of the trabecular bone density was decreased by LPS, and the decrease was significantly restored by tectoligenin treatment (Fig. 8B).

Osteoclast-induced bone resorption and inhibition of bone formation result in bone loss. The present inventors first confirmed the effect and mechanism of tectorigenin on osteoblast or osteoclast differentiation.

According to the experimental results, tectorigenin induced bone formation and mineralization potential on prematurely cultured osteoblasts without cytotoxicity.

Runx2 and osteerix are the major transcription factors required for activation of osteoblast differentiation and are essential for the regulation of genes involved in the production of bone-specific proteins. Tectorigenin upregulated Runx2 and osteogenic mRNA expression in primary cultured osteoblasts, suggesting that tectorigenin could activate the Runx2 pathway and regulate osteoblast differentiation.

In addition, periodontal ligament cells (PDLC) potentially differentiate into osteoblast-like cells in vitro and participate in bone healing in vivo . In addition, periodontal regeneration can be induced by selective cell migration, proliferation and differentiation of PDL cells and bone tissue.

Tectorigenin can increase ALP activity, calcified nodule formation, and mRNA levels of various markers to promote differentiation of osteoblasts in periodontal ligament cells, and the results suggest that tectorigenin is involved in a wide range of cells involved in bone regeneration This could be an impact.

BMP and MAPK pathways play an important role in in vitro osteoblast differentiation and in vivo bone remodeling.

Tectorigenin not only increases the expression of Bmp2, Bmp4 and Smad4 mRNA but also increases phosphorylation of Smad1 / 5/8, the central molecule on the BMP signal pathway, and the expression of Runx2 and Austerix, downstream target genes of the BMP signaling pathway . In addition, tectorigenin activated the ERK and JNK signaling pathways.

These results suggest that tectorigenin can activate BMP and MAPK signaling pathways and regulate osteoblast differentiation.

On the other hand, osteoclasts are involved in bone lysis in some bone diseases such as arthritis and periodontitis. Natural substances that inhibit the differentiation of osteoclasts are used therapeutically for the treatment of bone diseases including osteoclasts.

The present inventors have confirmed that tectorigenin can inhibit the differentiation of BMM precursor cells into TRAP-positive multinuclear osteoclasts. In addition, tectorigenin down-regulated the well known markers of osteoclast differentiation, TRAP, cathepsin-K and MMP-9 mRNA.

RANKL binds to RANK receptors expressed on osteoclast precursors, activating a variety of signaling pathways including the MAPK pathway, and regulating transcription factors such as c-Fos and NFATc1.

Tectorigenin reduced the expression levels of transcription factors, c-Fos and NFATc1, associated with osteoclastogenesis in RANKL-stimulated BMM osteoclast precursor cells. In addition, tectorigenin inhibited phosphorylation of p38 and ERK MAPKs induced by RANKL in BMM.

These results suggest that tectorigenin can down-regulate RANKL-induced transcription factor and MAPK to inhibit osteoclastogenesis.

Lipopolysaccharide (LPS), on the other hand, is widely known as a bone resorption inducer and displays molecular signals through TRAF6. In in vivo experiments with the LPS administration the mice, Tech storage genin is significantly reduced the bone resorption induced by LPS.

These results suggest that tectorigenin affects osteoclast differentiation by RANKL and can be used for the prevention or treatment of diseases related to bone loss.

That is, tectorigenin promotes osteogenic differentiation and bone regeneration through BMP and MAPK pathways, and inhibits RANKL-induced expression of MAPK, c-Fos and NFATc1, thereby reducing osteoclastogenesis. Thus, tectorigenin can be useful not only for the regeneration of bone tissue or periodontal tissue, but also for the treatment of bone-destructive diseases.

In addition, Runx2 gene activity has been known to be closely related to periodontal disease as well as bone formation promotion, and Runx2 gene has been reported to regulate alveolar bone remodeling and play an important role in maintaining periodontal ligaments (Camilleri, S. et al., Eur J Oral Sci., 114 (5): 361-373, 2006).

Therefore, the composition for promoting osteogenesis including tectorigenin can be useful not only for bone diseases but also for periodontal disease treatment by activating Runx2.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

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

Claims (11)

1. A composition for promoting osteogenesis comprising a compound of the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]

The method according to claim 1,
The compound of Formula 1 has Iris domestica (Belamcanda chinensis &lt; / RTI &gt; extract. The method according to claim 1,
A composition for promoting osteoblast differentiation and inhibiting osteoclast differentiation. The method of claim 3,
A composition for promoting bone formation that activates a BMP signaling pathway. 5. The method of claim 4,
Smad1, Smad5, and Smad8. &Lt; / RTI &gt; 5. The method of claim 4,
A composition for promoting osteogenesis that upregulates the expression of Runx2 and osterix. 5. The method of claim 4,
Wherein the phosphorylation level of ERK or JNK is increased to activate the BMP signal pathway. The method of claim 3,
A composition for promoting osteogenesis inhibiting the RANK signaling pathway. 9. The method of claim 8,
wherein the expression of c-Fos or NFATc1 is down-regulated. The method of claim 3,
A composition for promoting osteogenesis for the prevention or treatment of bone diseases. 11. The method of claim 10,
Wherein the bone disease is selected from the group consisting of osteoporosis, osteogenesis imperfecta, osteomalacia, osteopenia, fracture, bone defect, and hip arthropathy.

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