KR102014241B1 - Composition comprising sappanone A for inhibiting of bone loss - Google Patents

Abstract

The present invention relates to a composition for inhibiting bone loss containing sappanone A as an active component. By confirming that sappanone A significantly inhibits osteoclast differentiation and excessive bone absorption by osteoclasts, it is expected that sappanone A of the present invention can be usefully used for developing medicines for preventing or treating diseases related to bone loss or for developing health functional foods for alleviating diseases related to bone loss.

Description

Composition comprising sappanone A for inhibiting of bone loss}

The present invention relates to a composition for inhibiting bone loss comprising sapanon A as an active ingredient, and more particularly, a pharmaceutical composition or bone loss for preventing or treating a disease related to bone loss comprising sapanone A as an active ingredient. It relates to a dietary supplement for improving related diseases.

Recently, degenerative diseases have increased rapidly due to the increase in the average life span. In particular, bone health is closely related to physical activity of the elderly, contributing to maintaining physical health and maintaining quality of life, as well as suppressing secondary diseases that can occur when physical activity is suppressed. It is closely related.

Bone is a dynamic tissue that is destroyed and absorbed by osteoclasts and continuously produced by osteoblasts. Therefore, the balance between osteoclasts and osteoblasts is important to maintain bone homeostasis and health, and this breakdown of the balance causes bone disease (Raggatt L.J., et al., 2010). Therefore, the method for the prevention and treatment of bone diseases is to accelerate the bone production rate of osteoblasts or to inhibit the bone resorption by osteoclasts.

In particular, the balance disruption between osteoclasts and osteoblasts due to aging is caused by the breakdown of homeostasis due to excessive osteoclasting ability of osteoclasts rather than osteoblastic ability, which is a method of preventing and treating bone diseases caused by aging. It is important to inhibit bone uptake by cells (Tanaka, Y., et al., 2005).

Osteoclasts are produced by macrophage macrophages derived from hematopoietic stem cells through several differentiation steps by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (ANKL) (Asagiri, M. , et al., 2007; Chambers, TJ, 2000). When RANKL binds to RANK (receptor activator of nuclear factor-κB) on the surface of progenitor cells of osteoclasts, TRAF6 (TNF receptor associated factor 6), c-Fos and NFATc1 (nuclear factor of activated T cell c1) Activated to induce differentiation into osteoclasts (Sakae T., et al., 2003). In particular, the induction of TRAF6 was induced by mitogen-activated protein kinases (MAPKs), such as p38, extracellular-signal regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and nuclear factor-κB (NF-κB), and c- It activates factors important for differentiation of osteoclasts such as Fos and NFATc1 (Dickson, KM, et al., 2004). NFATc1 is an important transcription factor essential for osteoclast differentiation.Tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), cathepsin K (CtsK), matrix methalloproteinase 9 (MMP-9) It stimulates the expression of DC-STAMP (dendritic cell-specific transmembrane protein) and activates the differentiation and function of osteoclasts (Qing Hong, Z., et al., 2013).

In general, there are many advantages in finding new active ingredients from natural medicines used in traditional medicine, rather than experimentally modifying existing drugs to develop new ingredients. Recently, many studies have been conducted to develop or prevent the treatment of bone diseases caused by excessive bone resorption using many active compounds derived from natural products that have the effect of inhibiting osteoclast differentiation and function (An, J., et. al., 2016)

Sappanone A (SPNA) is a homoisoflavanone compound isolated from the heartwood of Caesalpinia sappan and has various pharmacological activities such as melanogenesis inhibition and anti-inflammatory activity. (Chang, TS, et al., 2012; Lee, S., et al., 2015; Liu, X., et al., 2016).

Thus, the present inventors found that in the course of studying bone disease treatment using an active compound derived from natural products, the sapanone A compound isolated from joiner inhibits the differentiation of osteoclasts and prevents excessive bone absorption by osteoclasts. The present invention was completed by confirming.

In the prior art, Korean Patent Publication No. 2013-0060834 describes the effect of treating the osteoporosis and rheumatoid arthritis of the compound isolated from the joiner extract, but the compound is a braziline compound, the composition of which is different from the sapanone A of the present invention. There is. In addition, Korean Patent Publication No. 2016-0099792 discloses a therapeutic effect of rheumatoid arthritis of a sapakalcon compound isolated from a joiner extract, but the sapanone A compound of the present invention is not described and mentioned. Korean Patent No. 0750657 discloses the effect of treating the inflammatory diseases of the joiner extract and the compounds isolated therefrom, but the bone loss inhibitory effect of sapanone A of the present invention is not described and mentioned at all. In Korean Patent No. 1704410, sapanone A isolated from a joiner extract is described, but the inhibition of osteoclast differentiation and bone resorption of sapanone A of the present invention is not described or implied at all.

Korean Unexamined Patent Publication No. 2013-0060834, a pharmaceutical composition having an osteoporosis inhibitory effect containing braziline or a pharmaceutically acceptable salt thereof as an active ingredient, published June 10, 2013. Korean Unexamined Patent Publication No. 2016-0099792, Composition for preventing or treating rheumatoid arthritis, including Sapanchalcone, 2016. 08. 23. Publication. Korean Registered Patent No. 0750657, Use of Joiner Extract and Compound Separated from It, 2007. 08. 13. Registered. Korea Patent No. 1704410, Composition for the prevention and treatment of cardiovascular diseases containing sapanone A, 2017. 02. 02. Registered.

An, J., et al., Natural products for treatment of bone erosive disease: the effects and mechanisms on inhibiting osteoclastogenesis and bone resorption, Int. Immunopharmocol., 36, 118-131, 2016. Asagiri, M., et al., The molecular understanding of osteoclast differentiation, Bone, 40, 251-264, 2007. Chambers, T. J., Regulation of the differentiation and function of osteoclasts, J. Pathol., 192, 4-13, 2000. Chang, T.S., et al., Melanogenesis inhibition by homoisoflavanone sappanone A from Caesalpinia sappan, Int. J. Mol. Sci., 13, 10359-10367, 2012. Dickson, K.M., et al., TRAF6-Dependent NF-κB Transcriptional Activity During Mouse Development, Dev. Dyn., 231, 122-127, 2004. Lee, S., et al., Sappanone A exhibits anti-inflammatory effects via modulation of Nrf2 and NF-κB, Int. Immunopharmacol., 28, 328-336, 2015. Liu, X., et al., Sappanone A attenuates allergic airway inflammation in ovalbumin-induced asthma, Int. Arch. Allergy Immunol., 170, 180-186, 2016. Qing Hong, Z., et al., The effect of rotative stress on CAII, FAS, FASL, OSCAR, and TRAP gene expression in osteoclasts, J. Cell Biochem., 114, 388-397, 2013. Raggatt, L. J., et al., Cellular and molecular mechanisms of bone remodeling, J. Biol. Chem., 285, 25103-25108, 2010. Sakae, T., et al., Signal transduction pathway regulating osteoclast differentiation and function, J. Bone Miner. Metab., 21, 123-133, 2003. Tanaka, Y., et al., Osteoblasts and osteoclasts in bone remodeling and inflammation, Curr. Drug Targets Inflamm. Allergy, 4, 325-328, 2005.

It is an object of the present invention to provide a composition for inhibiting bone loss comprising sapanon A (sappanone A) as an active ingredient.

The present invention relates to a composition for inhibiting bone loss comprising sapanone A of formula 1 as an active ingredient.

[Formula 1]

The sapanone A can inhibit the differentiation of osteoclasts.

The sapanone A can inhibit the bone resorption of osteoclasts.

The present invention also relates to a pharmaceutical composition for preventing or treating bone diseases, including the composition for inhibiting bone loss and a pharmaceutically acceptable excipient.

The present invention also relates to a health functional food for bone disease improvement comprising the composition for inhibiting bone loss and food supplements.

The bone diseases include osteoporosis, rheumatoid arthritis, osteopenia, periodontitis, periodontitis, osteolytic metastasis, osteoarthritis, senile kyphosis and It may be selected from the group consisting of paget disease.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for inhibiting bone loss comprising sapanone A of formula 1 as an active ingredient.

[Formula 1]

The sapanone A may be synthesized according to a conventional method, and may be prepared as a pharmaceutically acceptable salt. It may also be isolated and purified from the extract of Caesalpinia sappan L ..

The joiner extract may be extracted by adding one or more solvents selected from the group consisting of water, lower alcohols of C1 to C4, acetone, n-hexane, dichloromethane and ethyl acetate to joiner. The C1 to C4 lower alcohol may be methanol, propanol, isopropanol, butanol and isobutanol.

Sapanone A isolated from the joiner extract can be purified using column chromatography. The chromatography may include silica gel column chromatography, HP-20 column chromatography, RP-18 column chromatography, and LH-20 column chromatography. LH-20 column chromatography, high-performance liquid chromatography, and reverse-phase column chromatography may be used.

The "bone loss" may appear when the balance between osteoclast cells responsible for destruction and absorption of bone and osteoblasts responsible for bone formation is broken, and bone destruction and absorption mainly due to increased activation and formation of osteoclasts are excessive. May appear.

The sapanone A may inhibit bone loss by inhibiting differentiation and osteoresorption of osteoclasts from osteoclasts to osteoclasts.

In addition, the present invention relates to a pharmaceutical composition for preventing and treating bone diseases, including sapanone A of Formula 1 and a pharmaceutically acceptable excipient.

The sapanone A is preferably added in an amount of 0.001% to 50% by weight, more preferably 0.001% to 40% by weight, most preferably 0.001% to 30% by weight based on the total weight of the entire pharmaceutical composition. Can be.

The pharmaceutical composition may be used in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, and sterile injectable solutions according to conventional methods. Carriers, excipients and diluents that may be included in the pharmaceutical composition 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. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween-61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. More preferred dosage is 0.1 mg / kg / day to 500 mg / kg / day. Administration may be once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, abdominal, muscle, subcutaneous, intrauterine dural or cerebrovascular injections. In addition, since the pharmaceutical composition of the present invention is a composition derived from a natural product, there is almost no toxicity and side effects, and thus it is a drug that can be used safely even when taken for a long time for prevention purposes.

In addition, the present invention relates to a health functional food for improving bone disease, including sapanone A of the formula (1) and a food supplement acceptable food supplement.

The sapanone A is preferably added in an amount of 0.001% by weight to 50% by weight, more preferably 0.001% by weight to 30% by weight, most preferably 0.001% by weight to 10% by weight, based on the total weight of the health functional food. Can be.

The health functional food of the present invention includes the form of tablets, capsules, pills or liquids, and the food to which the extract of the present invention can be added, for example, various foods, beverages, gums, teas, vitamin complexes And health functional foods.

The bone disease may be a bone loss-related disease, the bone loss-related disease may be osteoporosis, rheumatoid arthritis, osteoopenia, periodontitis, periodontitis, osteolytic metastasis, Osteoarthritis, senile kyphosis and paget disease may be selected from, but is not limited thereto.

The present invention relates to a composition for inhibiting bone loss including sapanon A as an active ingredient, and it was confirmed that sapanone A significantly inhibits osteoclast differentiation and excessive bone uptake by osteoclasts.

Through this, it is expected that sapanone A of the present invention can be usefully used for the development of medicines for preventing or treating diseases related to bone loss or for improving functional foods for improving diseases related to bone loss.

Figure 1 shows the results of confirming the cytotoxicity of Sapanon A.
Figure 2 shows the results confirming the differentiation inhibitory effect of Sapanon A into osteoclasts induced by RANKL, (A) is a TRAP stained cell image, an indicator of osteoclasts, (B) is TRAP stained The graph shows the number of osteoclasts.
Figure 3 is induced by RANKL through the confirmation of mRNA expression inhibition of osteoclast markers CtsK (A), TRAP (B), DC-STAMP (C), MMP-9 (D) and OSCAR (E) of Sapanone A It shows the result of confirming the effect of inhibiting the differentiation into osteoclasts.
Figure 4 shows the results confirming the degree of activation (phosphorylation) inhibition of signaling agents related to osteoclast differentiation of sapanone A.
FIG. 5 shows the inhibition of the activation (phosphorylation) of Akt and GSK-3β (A) and the expression of NFATc1, a major transcription factor related to osteoclast formation, among the osteoclast differentiation-related signal transduction agents of sapanone A (B ) Shows the result of confirming the effect.
Figure 6 shows the results confirming the effect of inhibiting the actin-ring formation (actin-ring formation) by RANKL of Sapanone A, (A) is a graph showing the actin ring formation image, (B) is a graph Is showing.
7 shows the results of confirming the inhibition of bone absorption of sapanone A, (A) shows the results of the surface of the absorption pit (resorption pit), (B) shows the results of the graph quantifying the degree of bone absorption Giving.
Figure 8 confirms the effect of inhibiting bone loss of sapanone A in the LPS-induced bone loss mouse model, (A) shows a three-dimensional reconstruction of the femur image by scanning the left femur with a high-resolution micro CT analyzer, (B) is a graph showing quantitative bone volume (BV / TV) per tissue volume of the left mouse femur, (C) the number of trabecular tissues (Tb.N), (D) the trabecular separation (Tb.SP) Is showing.
Figure 9 shows the results of confirming the number of osteoclasts in the left femur tissue of the LPS-induced bone loss mouse model, (A) of the control mice not treated anything, (B) of the mice treated with LPS, ( C) shows the results of staining osteoclasts in the left femur tissue of mice treated with LPS and sapanone A.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the information provided herein is to be thorough and complete, and to fully convey the spirit of the present invention to those skilled in the art.

Example 1 Separation of Sapanone A Compound

Sappanone A used in the present invention was isolated and purified from the extract obtained from the heartwood of joiner.

10 kg of seedlings were added to 3 liters of methanol in a reflux apparatus, followed by treatment for 3 hours to obtain a joiner extract. The obtained extract was removed from the solvent in a manner of lowering atmospheric pressure, and a residue was obtained. The obtained residue was suspended in water (3,000 ml) and then partitioned between n-hexane (9,000 ml), ethyl acetate (EtOAc) (9,000 ml) and n-butanol (n-BuOH) (9,000 ml). 960 g of the EtOAc fraction was fractionated by silica gel column chromatography (column size: 200 × 700 mm) according to the concentration gradient elution condition of chloroform: methanol (50: 1 → 0: 1 [v: v]) to obtain 14 fractions. (Fr.1-Fr.14). Fr. 5 (4.4 g) was fractionated by reverse phase column chromatography using methanol: water (2: 1 → 0: 1 [v: v]) gradient elution to obtain 7 small fractions (Fr.5-1- Fr.5-7). High-performance liquid chromatography (Semipreparative HPLC, YMC Pak ODS-A column, column) of Fr.5-3 (728 mg) in the small fraction according to isocratic elution conditions of a 30% methanol and 0.1% trifluoroacetic acid (TFA) mixed solution Sapanon A compound was separated by size: 20 × 250 mm, particle size: 5 μm, UV detection: 210 nm, flow rate: 5 mL / min.

Example 2 Confirmation of Physicochemical Structure of Sapanone A

Sappanone A;

SPNA;

White amorphous powder;

EI-MS [M] ⁺ 284 m / z ;

¹ H NMR and ¹³ C NMR data see Table 1 below.

Position δ _H δ _c One 2 5.37 (2H, s) 69.2 3 127.8 4 183.2 4a 116.0 5 7.81 (1H, doublet, J = 8.4 Hz) 130.8 6 6.54 (1H, doublet, J = 8.8 Hz) 112.3 7 166.7 8 6.33 (1H, s) 103.7 8a 165.0 9 7.66 (1 H, s) 138.6 One' 129.6 2' 6.84 (1 H, s) 116.7 3 ' 148.9 4' 146.7 5 ' 6.88 (1H, doublet, J = 8 Hz) 118.5 6 ' 6.79 (1H, doublet, J = 8 Hz) 124.7

< Example  3. Animal Cell Culture and Sample Processing>

Example  3-1. Bone Marrow-derived Macrophage Isolation

Bone marrow-derived macrophages (bone marrow mcrophage, BMM) were isolated to induce differentiation of osteoclasts.

Bone marrow cells were isolated from the femur and tibia of male 6-year-old ICR mice, containing α culture containing 10% FBS (fetal bovine serum), 100 U / ml penicillin and 100 µg / ml streptomycin. -MEM (Minimum Essential Media (Hyclone, USA)) in a medium containing 10ng / ml macrophage colony-stimulating factor (M-CSF) was incubated overnight at 37 ℃, 5% CO ₂ cell incubator. After culturing, the cells floating in the culture were collected and put in a medium containing 30 ng / ml M-CSF in the basal culture medium and cultured for 3 days. After 3 days, the culture solution was removed, and the cells adhered to the plate were washed with PBS (phosphate buffered saline), and cells were collected using a scraper to separate bone marrow-derived macrophages.

Example 3-2. Induction of differentiation into osteoclasts and sample processing

Bone marrow-derived macrophages isolated in Example 3-1 were dispensed with 5 × 10 ⁴ cells per well into 96 well plates, and 30 ng / ml M-CSF and 100 ng / ml receptor activator of nuclear factor-κB ligand. ) Was added and cultured for 7 days to induce differentiation into osteoclasts. At this time, it was confirmed that the effect on the differentiation into osteoclasts by treating with Sapanon A (SPNA) together.

<Example 4. Confirmation of the effect of sapanone A on osteoclast differentiation>

Example 4-1. Cytotoxicity of Sapanone A

Prior to confirming the effect of sapanone A on osteoclast differentiation, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide to determine cytotoxicity by sapanone A Assays were performed.

The bone marrow-derived macrophages isolated in Example 3-1 were dispensed with 1 × 10 ⁴ cells per well in a 96-well plate, placed in a basic culture medium containing 30ng / ml M-CSF, and cultured for 72 hours. . At this time, sapanone A was treated together to be 0, 3, 10, 30 uM. After 72 hours, MTT solution was added to the cells at a concentration of 0.5 mg / ml, the reaction was carried out for 3 hours, the culture solution was removed, the formed formazan precipitate was dissolved in DMSO (dimethyl sulfoxide), and the absorbance was measured at 540 nm. The cell viability of the non-saponone A treated cells was calculated to be 100%, and the cell viability of each sapanone A treated concentration was calculated. The results are shown in FIG. 1.

As shown in Figure 1, the treatment of saponon A (SPNA) to bone marrow-derived macrophages did not reduce cell viability was confirmed.

Through this, it was found that the sapanone A of the present invention is not cytotoxic.

Example 4-2. Confirmation of osteoclast differentiation using TRAP staining

Differentiation into osteoclasts was confirmed through staining of TRAP (tartrate-resistant acid phosphatase), an indicator of osteoclasts.

Sapanone A of Example 3-2 was treated by concentration, and the cells induced with differentiation into osteoclasts were fixed by treating with 3.7% formalin for 15 minutes, and 0.1% Triton X-100 (triton X-100) was used. After treatment to increase cell permeability, cells stained reddish purple through TRAP staining were determined as osteoclasts, and the results are shown in FIG. 2.

As shown in the graph (S) of the osteoclast image stained by TRAP staining (A) and the number of the osteoclasts stained in FIG. 2, when the saponon A was not treated, the osteoclasts stained with reddish purple were many. On the other hand, in the case of treatment with sapanon A, it was confirmed that the number of osteoclasts stained in reddish purple according to the treatment concentration.

Through this, it can be seen that sapanone A of the present invention inhibits osteoclast differentiation in a concentration-dependent manner.

Example 4-3. Identifying osteoclast differentiation using osteoclast markers

To determine the differentiation of osteoclasts, tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), cathepsin K (CtsK), matrix methalloproteinase 9 (MMP-9), and DC-STAMP ( mRNA levels of dendritic cell-specific transmembrane proteins were determined.

Induction of osteoclast differentiation was performed in the same manner as in Example 3-2. At this time, Sapanone A was treated to be 0, 3, 10, and 30 uM, and 100ng / ml of RANLK was treated for 7 days to induce osteoclast differentiation, and cells not treated with Sapanone A and RANLK were used as a control. It was. The cultured cells were collected and total RNA was isolated using a manual provided by RNeasy Mini Kit (QIAGEN, USA) and company. CDNA was synthesized using 1 μg of isolated RNA and RT-PCR (reverse transcription polymerase chain reaction) kit (Invitrogen, USA). Using the synthesized cDNA as a template, qPCR (real-time quantitative PCR) was performed using the primers of Table 2 and TOPreal qPCR 2 × PreMIX (Enzynomics, KOR) to confirm the mRNA expression levels of osteoclast markers. The results are shown in FIG. At this time, β-actin (β-actin) was used as a quantitative control for mRNA expression analysis.

Gene Primer type Primer Sequence MMP-9 sense 5′-TGG GCA AGC AGT ACT CTT CC-3 ′ antisense 5′-AAC AGG CTG TAC CCT TGG TC-3 ′ CtsK sense 5′-GAC ACC CAG TGG GAG CTA TG-3 ′ antisense 5′-AGA GGC CTC CAG GTT ATG GG-3 ′ TRAP sense 5′-ACT TGC GAC CAT TGT TAG CC-3 ′ antisense 5′-TTC GTT GAT GTC GCA CAG AG-3 ′ β-actin sense 5′-GGG AAA TCG TGC GTG ACA TCA AAG-3 ′ antisense 5′-AAC CGC TCC TTG CCA ATA GT-3 ′ OSCR and DC-STAMP are purchased by using primers from Origene (USA)

As shown in FIG. 3, when only RANKL is treated, CtsK (A), TRAP (B), DC-STAMP (C), While both mRNA expression levels of MMP-9 (D) and OSCAR (E) were increased, it was confirmed that mRNA expression levels of all osteoclast markers increased by RANKL decreased significantly with sapanone A treatment concentration.

Through this, it was found that the sapanone A of the present invention inhibits osteoclast differentiation induced by RANKL.

Therefore, it can be seen from the above results that the sapanone A of the present invention is not cytotoxic and has an excellent inhibitory effect on the differentiation of osteoclasts.

<Example 5. Confirmation of the influence of sapanone A on the mechanism of osteoclast differentiation>

Example 5-1. Confirmation of Sapanone A's Effect on Osteoclast Differentiation-related Signal Transduction Mechanism

RANKL binds to RANK present on the surface of progenitor cells of osteoclasts, and the signaling mechanisms such as NF-κB, PI3K (phosphatidylinositol 3-kinase) / AKT, and MAPK are phosphorylated to activate and differentiate osteoclasts. Therefore, the degree of phosphorylation of each signal transduction material was confirmed by Western blot in order to confirm the effect of the saponon A of the present invention on signaling mechanisms related to osteoclast differentiation.

Induction of osteoclast differentiation was performed in the same manner as in Example 3-2. At this time, the cells treated with 30 uM sapanon A and the untreated cells were divided, 100 ng / ml RANKL was added to all wells, and treated for 0, 15, 30, and 60 minutes, and the cells were collected. Western blots were performed using the proteins obtained by lysis of the collected cells, and the results are shown in FIG. 4. At this time, p38, ERK, JNK, and AKT were selected as signaling materials related to osteoclast differentiation, and each signaling material and their phosphorylated forms (p-p38, p-ERK, p-JNK, p-AKT) were selected. Was treated with an antibody capable of detecting.

As shown in Figure 4, the degree of phosphorylation of p38, ERK, JNK was confirmed that there is no difference according to the treatment with sapanon A, whereas in the case of AKT, phosphorylation of ANKT induced by RANKL about 30 minutes after sapanone A treatment It was confirmed that is suppressed.

Example  5-2. Sapanon  A's AKT Signal transduction To mechanism  Check impact

In order to specifically confirm the phosphorylation inhibitory effect of AKT related to osteoclast differentiation of sapanone A identified in Example 5-1, the phosphorylation of GSK-3β, an AKT subsignaling agent, and osteoclast formation below The expression of NFATc1, a major transcription factor, was confirmed by Western blot.

Proceed in the same manner as in Example 5-1, and treated with 0, 10, 30uM sapanone A for 30 minutes to confirm the phosphorylation of AKT and GSK-3β. To confirm the expression of NFATc1, 30 uM of sapanone A was treated and differentiation was induced for 24 or 48 hours. After each treatment time, the cells were collected and lysed, and Western blot was performed using the obtained protein. The results are shown in FIG. 5. At this time, the antibody to AKT, p-AKT, GSK-3β, p-GSK-3β or NFATc1 was treated.

As can be seen from the results of confirming the degree of phosphorylation of AKT and GSK-3β in FIG. 5 (A), it was confirmed that phosphorylation of AKT and GSK-3β is reduced according to the concentration of sapanone A.

In addition, as shown in the results of confirming the expression level of NFATc1 in FIG. It was confirmed to suppress.

Therefore, through the above results, the osteoclasts of the present invention inhibit the expression of AKT and GSK-3β signaling agents and inhibit the expression of NFATc1, an important transcription factor for osteoclast formation through the inhibition of osteoclasts. It was found to inhibit differentiation and formation.

<Example 6. Confirmation of the effect of inhibiting bone absorption of sapanone A>

Example 6-1. Confirmation of actin-ring formation inhibitory effect

Actin-ring formation is an important indicator of bone resorption activity of osteoclasts and is the most obvious feature of osteoclasts matured during osteoclastogenesis. Thus, the degree of actin ring formation was confirmed in order to confirm whether the spanone A of the present invention inhibits bone absorption.

Bone marrow-derived macrophages isolated in Example 3-1 were dispensed on a plate covered with a cover glass, and cultured for 7 days in a basic culture medium containing 30ng / ml M-CSF and 100ng / ml RANKL. To induce differentiation into osteoclasts. At this time, saponone A was treated with different concentrations (0, 3, 10, 30 uM) to induce differentiation of osteoclasts. After 7 days, the cells were washed with PBS, treated with 4% paraformaldehyde for 15 minutes, immobilized, and treated with 0.1% Triton X-100 to increase cell permeability. FITC-phalloidin) was treated in the cow for 10 minutes. After washing the cells with PBS, the cells were mounted and observed for actin ring formation using a Zeiss LSM510 META NLO inverted confocal laser scanning microscope (Zeiss, Germany), and the results are shown in FIG. 6.

As shown in the results of analyzing the microscopic image of FIG. 6 (A) and the number of actin rings formed in FIG. 6 (B), in the case where the sapanone A was not treated, a large number of actin rings were formed. In the case of treatment, it was confirmed that the number of actin rings was reduced according to the treatment concentration.

Through this, it was found that sapanone A of the present invention inhibits the bone resorption function of osteoclasts.

Example 6-2. Confirmation of Bone Absorption Effect

A bone resorption assay was performed to confirm the effect of inhibiting bone absorption of sapanone A of the present invention.

The bone marrow-derived macrophages isolated in Example 3-1 were dispensed with 5 × 10 ⁴ cells per well in Corning's OsteoAssay Surface 96 well plate (Corning, USA), 30ng / ml M-CSF and 100ng / ml RANKL Put the basic culture medium containing was incubated for 7 days to induce differentiation into osteoclasts. At this time, saponone A was treated with different concentrations (0, 3, 10, 30 uM) to induce differentiation of osteoclasts. After 7 days, washed with tap water and observed the surface of the absorption pit using H550 Nikon Microscope, quantified the degree of bone absorption using the Image J (Image J) program, the results are shown in Figure 7 .

As shown in the absorbent pit surface image of FIG. 7 (A) and the graph analyzing the degree of bone uptake of FIG. 7 (B), when the saponone A was not treated, compared to the ratio of the bone absorption pit by the osteoclast, When A was treated, it was confirmed that the ratio of bone absorption pits decreased with treatment concentration.

Through this, it was found that the sapanone A of the present invention inhibits the bone resorption of osteoclasts.

Therefore, the above results, it was found that the sapanone A of the present invention prevents bone loss by inhibiting the bone resorption function of osteoclasts.

<Example 7. Confirmation of the bone loss inhibitory effect of Sapanone A>

In order to confirm the bone loss inhibitory effect of sapanone A, a LPS (lipopolysaccharide) -induced bone loss mouse model was prepared.

Six-week-old male mice were randomly divided into four groups of four per group and the samples were treated according to Table 3 below. Sapanone A was injected intraperitoneally 1 hour before the first LPS injection at a concentration of 50 mg / kg and intraperitoneally for 8 days every other day. LPS was injected intraperitoneally on days 2 and 6 at a concentration of 5 mg / kg. On day 9 all mice were sacrificed and the left femoral defect site was scanned using a high resolution micro CT analyzer. After three-dimensional reconstruction using scan files, bone volume (BV / TV), trabecular separation (Tb.SP) and trabecular number (Tb.N) per tissue volume Quantitation was performed using INFINITT-Xelis software, and the results are shown in FIG. 8.

In addition, after removing the calcareous in the left femur of the mouse, the osteoclasts were observed by TRAP staining, the results are shown in FIG.

group Sample processing Group-1 No treatment Group-2 Sapanon A Group-3 LPS Group-4 LPS + Sapanone A

In the mouse femur image results of FIG. 8 (A), intratramural trabeular was reduced by LPS treatment, and when sapanon A (SPNA) was treated, it was confirmed that the reduction of the reduced trabecular strain by LPS treatment was suppressed. It was.

Also, for the bone volume per tissue volume (BV / TV) of FIG. 8 (B) and the number of trabecular tissues (Tb.N) of FIG. 8 (C), the bone volume per tissue volume (BV / TV) reduced by LPS and It was confirmed that the number of fibers was increased by Sapanone A. On the other hand, in the case of the fibrous column separation (Tb.SP) of Figure 8 (D) it was confirmed that the increase by the LPS treatment is reduced by the sapanon A treatment.

As shown in the results of Figure 9, compared to the control group was not treated (A) it was confirmed that there is a significant presence of osteoclasts in the mouse femur bone loss induced by LPS (B). On the other hand, when treated with LPS and Sapanon A, it was confirmed that the number of osteoclasts in the femur increased by LPS was reduced by treating Sapanone A (C).

Through this, it can be seen that sapanone A of the present invention inhibits bone loss due to bone resorption by osteoclasts induced by LPS.

Therefore, it was found that sapanone A of the present invention is excellent in inhibiting bone loss and can be usefully used for the treatment of diseases related to bone loss.

Preparation Example 1. Pharmaceutical Formulations

Formulation Example 1-1. Manufacture of tablets

200 g of sapanone A was mixed with 175.9 g lactose, 180 g potato starch and 32 g colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

Formulation Example 1-2. Preparation of Injection

1 g of sapanone A, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

Preparation Example 2 Preparation of Health Functional Food

Formulation Example 2-1. Preparation of health functional food

20 g of sapanone A of the present invention, a vitamin mixture amount, 70 μg of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 μg of vitamin B12, vitamin C 10 mg, biotin 10 Μg, nicotinic acid amide 1.7 mg, folic acid 50 µg, pantothenate calcium 0.5 mg, mineral mixture appropriate amount, ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium monophosphate 15 mg, calcium diphosphate 55 mg , Potassium citrate 90mg, calcium carbonate 100mg, magnesium chloride 24.8mg mixed to prepare a granule, but can be prepared by modifying the formulation in a variety of formulations. In addition, the composition ratio of the above-mentioned vitamin and mineral mixture may be arbitrarily modified, and it may be prepared by mixing the above components according to a conventional health functional food manufacturing method.

Formulation Example 2-2. Preparation of Health Functional Drink

1g of the present invention, citric acid 0.1g, fructooligosaccharide 100g, purified water 900g was mixed, stirring, heating, filtration, sterilization, refrigeration according to the conventional beverage production method to prepare a beverage.

Claims (7)

A composition for inhibiting bone loss, including sapanone A of formula 1 as an active ingredient.
[Formula 1]

The method of claim 1,
Said sapanone A is a composition for inhibiting bone loss, characterized in that to inhibit the differentiation (differentiation) of osteoclasts. The method of claim 1,
Said sapanone A is a composition for inhibiting bone loss, characterized in that it inhibits bone resorption of osteoclasts. Osteoporosis, osteoopenia, osteolytic metastasis, characterized in that it comprises a composition for inhibiting bone loss of any one of claims 1 to 3 and a pharmaceutically acceptable excipient, A pharmaceutical composition for preventing or treating bone diseases selected from the group consisting of senile kyphosis and paget disease. delete Osteoporosis, osteoopenia, osteolytic metastasis, characterized in that it comprises a composition for inhibiting bone loss according to any one of claims 1 to 3 and a food-acceptable food supplement additive. ), Health functional food for improving bone disease selected from the group consisting of senile kyphosis and paget disease. delete

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