COMPOSITION COMPRISING OSMUNDACETONE OR PHARMACEUTICALLY ACCEPTABLE SALT THEREOF FOR PREVENTING OR TREATING BONE DISEASE

Abstract

The present invention relates to a composition comprising osmundacetone or a pharmaceutically acceptable salt thereof for preventing or treating bone diseases. More specifically, the present invention relates to: a composition comprising osmundacetone, a pharmaceutically acceptable salt thereof, or an Osmunda japonica extract as an effective ingredient, for preventing or treating osteoporosis, rheumatoid arthritis, arthralgia, Paget disease, bone metastatic cancer, or fracture; a food composition for improvement; a use of a salt; and a treatment method. The composition according to the present invention shows a strong inhibitory activity against proliferation and differentiation of osteoclast which causes bone loss, and activation of differentiation of osteoblast, and thus can be usefully utilized in developing medicines for safe and effective treatment of bone diseases or functional foods for improving symptoms of bone diseases

Claims

1-9. (canceled)

10. A method for treating a bone disease in a subject, the method comprising administering an effective amount of a composition comprising osmundacetone or a pharmaceutically acceptable salt thereof as an active ingredient to a subject in need thereof, wherein the bone disease is at least one selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastatic cancer, and bone fractures.

11-12. (canceled)

13. A method for treating a bone disease in a subject, the method comprising administering an effective amount of a composition comprising an Osmunda japonica extract as an active ingredient to a subject in need thereof, wherein the bone disease is at least one selected from the group consisting of osteoporosis, rheumatoid arthritis, arthralgia, Paget's disease, bone metastatic cancer, and bone fractures.

14. The method of claim 13, wherein the extract is extracted with at least one solvent selected from the group consisting of water, ethanol, grain ethanol, methanol, propanol, isopropanol, butanol, acetone, ether, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, petroleum ether, diethyl ether, and benzene.

15. The method of claim 10, wherein the composition is a pharmaceutical composition or a food composition.

16. The method of claim 10, wherein the osmundacetone is isolated from a plant in the family Osmundaceae.

17. The method of claim 16, wherein the plant in the family Osmundaceae is Osmunda japonica.

18. The method of claim 13, wherein the composition is a pharmaceutical composition or a food composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0095] FIG. 1 shows HPLC results of water (hot water) or ethyl acetate (EA) extract of Osmunda japonica in order to isolate and identify a substance having osteoclast differentiation inhibitory activity contained in Osmunda japonica (detection wavelength: 280 nm). EA-2 represents the second fraction among EA extracts separated as seven fractions. The peak of osmundacetone was marked by a black arrow. The peaks marked with red ellipses indicate the same peak position observed during a purification process.

[0096] FIG. 2 shows TRAP assay results using mouse bone marrow cells in order to investigate the activity of Osmunda japonica extracts, a fraction thereof, and osmundacetone to inhibit the proliferation and differentiation of osteoclasts.

[0097] FIG. 3 shows TRAP assay results using mouse bone marrow cells to investigate the osteoclast proliferation and inhibition inhibitory activity of 1, 4, 7, and 10 M osmundacetone commercially purchased (Alfa Aesar, Thermo Fisher Scientific) in order to obtain the osteoclast differentiation inhibitory ability, IC.sub.50, of osmundacetone.

[0098] FIG. 4A shows the results of confirming IC.sub.50 of osmundacetone and the known drug Fosamax after bone marrow cells were treated with various concentrations of osmundacetone and Fosamax to induce differentiation thereof, in order to compare the osteoclast differentiation inhibition effect between osmundacetone and Fosamax. FIG. 4B shows the results of confirming the ability of osmundacetone to inhibit osteoclast differentiation and activate osteoblast differentiation simultaneously while osteoclast precursor cells and osteoblast precursor cells were co-incubated.

[0099] FIG. 5 shows the results of confirming the expression of OCN by performing western blot after the treatment with osmundacetone, in order to investigate the effect of osmundacetone on the OCN production of osteoblasts.

[0100] FIG. 6 shows the results of confirming the expression of RUNX2 by performing western blot after the treatment with osmundacetone, in order to investigate the effect of osmundacetone on the expression of RUNX2.

MODE FOR CARRYING OUT THE INVENTION

[0101] Hereinafter, the present invention will be described in detail.

[0102] However, the following examples are merely for illustrating the present invention and are not intended to limit the scope of the present invention.

<Example 1> Experimental Methods

[0103] 1. Preparation of Osmunda japonica Extracts and Fractions and Isolation and Identification of Compound

[0104] Osmunda japonica (or Osmundae Rhizoma) was extracted with hot water and ethyl acetate sequentially, and then an ethyl acetate extract (EA extract) was separated into seven fractions through HPLC.

[0105] Specifically, as the Osmunda japonica extract, a hot-water extract obtained by clearly washing 200-250 g of Osmunda japonica collected in Gangwon-do, Korea, placing the washed Osmunda japonica in a steaming container (OSK-2002, Doctor Red Ginseng, Well Sosana, Daewoong Pharmaceutical Inc.), adding 1.5 L of water, steaming for 24 hours, further adding 3.5 L of water, aging for 72 hours, and refrigerating, was used. The same volume of ethyl acetate (EA) was added to the hot-water extract, followed by well mixing, and then the EA layer was dried using a rotary evaporator, and then used as an EA extract. The EA extract was dissolved in a minimum amount of DMSO, and then diluted with water, and here, as for a dilution factor, the EA extract was diluted to 70% of the original volume of the hot-water extract assuming a yield of approximately 70%. The EA extract was separated into seven fractions through HPLC. The second fraction was dried using a rotary evaporator, and then used as EA-2 extract. It was observed that among the seven fractions, the second fraction (EA-2) had osteoclast differentiation inhibitory activity (see FIG. 1, <Example 2>).

[0106] In order to identify a substance having osteoclast differentiation inhibitory activity, nine single compounds were isolated from the EA-2 fraction, followed by purification, and then the kind and chemical structure of each compound were established through nuclear magnetic resonance (NMR) and mass spectrometry (MS). The conditions of HPLC for isolating the single compounds from the EA-2 fraction are shown in Table 1. The substance showing osteoclast differentiation inhibitory activity among the isolated compounds was confirmed to be osmundacetone (see <Example 2>). Specific NMR and MS results of the osmundacetone identified from the Osmunda japonica extract are as follows:

[0107] .sup.1H-NMR (700 MHz, methanol-d4) 7.52 (1H, d, J=16.1, H-7), 7.08 (1H, d, J=2.1 Hz, H-2), 6.99 (1H, dd, J=7.7, 2.1 Hz, H-6), 6.79 (1H, d, J=7.7 Hz, H-5), 6.55 (1H, d, J=16.1 Hz, H-8), 2.34 (3H, s, H-10); .sup.13C-NMR (175 MHz, methanol-d4) 201.7 (C-9), 150.1 (C-4), 147.1 (C-3), 147.0 (C-7), 127.9 (C-1), 124.9 (C-8), 123.7 (C-6), 116.7 (C-5), 115.4 (C-2), 27.2 (C-10); ESI-MS (negative mode) m/z 177 [MH]; ESIMS (positive mode) m/z 179 [M+H]+, 201 [M+Na].sup.+.

TABLE-US-00001 TABLE 1 EA-2 fraction HPLC separation method HPLC Agilent 1200 Column Kromasil 100-5-C18 (4.6*250 mm) Detector UV (210 nm, 254 nm, 280 nm) Flow 1 ml/min Oven 30 C. Injection 10 l Mobile phase A (0.05% TFA in H.sub.2O) % B (MeOH) % 0 min 80 20 20 min 0 100 30 min 0 100

[0108] 2. Cell Culture

[0109] MC3T3-E1 subclone 4 murine osteoblastic cells, (# CRL-2593TM, ATCC) were incubated using -MEM (Minimum Essential Medium Eagle-Alpha Modification, # LM00853, WELGENE, Seoul, Korea) in the conditions of 37 C. and 5% CO.sub.2.

[0110] HaCaT human epidermal keratinocytes, # ATCC PCS-200-011) were incubated using Dulbecco's modified Eagle's medium (DMEM, # DMEM-HPA, Capricorn Scientific, Ebsdorfergrund, Germany) in the conditions of 37 C. and 5% CO.sub.2. NIH3T3 mouse embryo fibroblasts (# KCLB 21658), RAW264.7 murine macrophages (preosteoclast cell line, # KCLB 40071), HCT116 human colon cancer cells (# KCLB 10247), PC3 human prostate adenocarcinoma cells (# KCLB 21435), HT1080 human fibrosarcoma cells (# KCLB 1121), and B16F10 mouse malignant melanoma cells (# KCLB 80008) were purchased from the Korean Cell Line Bank, and incubated using DMEM in the conditions of 37 C. and 5% C02.

[0111] AGS human stomach adenocarcinoma cells (# KCLB 21739), A549 human lung carcinoma cells (# KCLB 10185), Caki-1 human kidney carcinoma cells (# KCLB 30046), T24 human bladder carcinoma cells (# KCLB 30004), TC-1 P3 HPV-16E7-expressing mouse pulmonary epithelial cells, MHC class I (provided by Tae-Woo Kim, a professor of Korea University) were incubated using RPMI (Rosewell Park Memorial Institute) 1640 (# RPMI-A, Capricorn Scientific, Ebsdorfergrund, Germany) in the conditions of 37 C. and 5% CO.sub.2.

[0112] Human adipose-derived mesenchymal stem cells, ADMSCs (CEFO Bio, Seoul, Korea), were incubated using CB-ADMSC-GM (CEFO Bio, Seoul, Korea) in the conditions of 37 C. and 5% CO.sub.2.

[0113] 3. Primary Culture of Osteoclasts

[0114] Bone marrow cells were collected from the femur and shin bones of 5-8 week old male C57BL/6 mice. Muscles were removed from the bones, and stored in cold phosphate buffered saline (PBS; # CAP08-050, GenDEPOT, Katy, Tex., USA).

[0115] Both ends of each bone were cut, and the bone marrow was flushed with a flushing medium (serum-free -MEM, 2 mM ethylenediaminetetraacetic acid) kept cool, using a 25 G needle.

[0116] Bone marrow cells were collected by centrifugation at 3,000 rpm for 3 min using Labogene 1248R (Labogene, Lynge, Denmark), and then resuspended in a wash medium. Thereafter, 8 mL of the collected bone marrow cells were overlaid on 6 mL of a lymphocyte isolation medium (LSM; #50494, MP Biomedicals, Santa Ana, Calif., USA), followed by centrifugation at 1,600 rpm for 20 minutes, thereby isolating mononuclear cells.

[0117] The mononuclear cell bands were collected from the media interface, and incubated using complete -MEM (containing 10% fatal bovine serum (FBS; Capricorn Scientific, Ebsdorfergrund, Germany) and 1% (v/v) antibiotic (including 100 U/mL penicillin G and 100 mg/mL streptomycin)) in the conditions of 37 C. and 5% CO.sub.2 while the medium was exchanged every three days.

[0118] In order to promote the differentiation of osteoclasts, 110.sup.5 cells were incubated in 0.5 mL of the medium per well in a 48-well plate, in the presence of osteoclast differentiation factors, M-CSF (60 ng/mL) and RANKL (150 ng/mL), purchased from PeproTec (Seoul, Korea). As known, macrophages/monocytes derived from one bone marrow differentiated into mature multinucleated osteoclasts after 6 days (Gurt et al., 2015).

[0119] 4. Co-Culture of Osteoclasts and Osteoblasts

[0120] 110.sup.5 C57BL/6 mouse bone marrow mononuclear cells per well of a 48-well plate were prepared in the same manner as described above, and co-incubated together with 1.510.sup.4 MC3T3-E1 murine osteoblasts, using 500 L of -MEM containing 10% FBS and 1% antibiotic (100 U/mL penicillin G and 100 mg/mL streptomycin) in the conditions of 37 C. and 5% CO.sub.2 while the medium was exchanged every three days.

[0121] Positive control cells were co-incubated together with osteoclast differentiation factors M-CSF (60 ng/mL) and RANKL (150 ng/mL), and osteoblast differentiation factors ascorbic acid (50 g/mL) and 10 mM R-glycerophosphate.

[0122] Negative control cells were incubated by adding only M-CSF as a differentiation factor. In order to examine effects of osmundacetone on the differentiation of osteoclasts and osteoblasts, osmundacetone were administered at a final concentration of 10 M into the positive control cells on day 1 after the cells were dispensed on the plate.

[0123] In the positive control group treated with osmundacetone, osteoclasts completely disappeared 6-7 days after cell dispensing, and thus while only osteoblast differentiation factors were administered, the cells were incubated from 8 days to 21 days after cell dispensing.

[0124] 5. Analysis of Bone Resorption

[0125] The quantitative measurement of in vitro osteoclast-mediated degradation of human bone collagen was carried out using the OsteoLyse Assay Kit (Lonza Walkersville, Inc. Walkersville, Md., USA) according to manufacturer's instructions.

[0126] This assay allows a direct measurement of the release of matrix metalloproteinase into the osteoclast resorption lacuna (Delaisse et al., 2003).

[0127] Briefly, 210.sup.4 mouse bone marrow cells prepared above were dispensed in 96-well OsteoLyse cell culture plate coated with europium-conjugated collagen.

[0128] The cells were incubated in 0.1 mL of complete -MEM per well in the presence of M-CSF (60 ng/mL) and RANKL (150 ng/mL) in the conditions of 37 C. and 5% CO.sub.2 for 6 days. Thereafter, the medium was exchanged on day 3 of the dispensing.

[0129] In order to measure the ability to inhibit the mature osteoclastic functions, the medium was exchanged on day 6, and osmundacetone was added at an IC.sub.50 concentration obtained from TRAP analysis.

[0130] After the mature osteoclasts were treated with osmundacetone for 3 days, 10 L of a supernatant of the cell culture was taken, and placed in a second 96-well analysis plate containing fluorophore-Releasing reagent. The degraded collagen was measured using a time-resolved fluorescence fluorimeter, Wallac Victor (Perkin Elmer, Waltham, Mass., USA). Here, the measurement was carried out using excitation at 340 nm and emission at 615 nm for a time interval of 400 s after an initial delay of 400 s.

[0131] The bone resorption rate (%) was obtained by calculating the proportion of the amount of bone resorption by the presence of osmundacetone compared with the non-treated control group, and was normalized by cellular DNA.

[0132] 6. Western Blot

[0133] In order to analyze the expression of osteoblast differentiation markers during a differentiation procedure, 310.sup.5 MC3T3-E1 cells were dispensed in a 100 mm culture plate together with 10 mL of complete -MEM containing ascorbic acid (50 g/mL) and 10 mM R-glycerophosphate per well. Here, 50 M osmundacetone was added or not added, and the cells were incubated at 37 in a 5% CO.sub.2 incubator for 21 days while the medium was exchanged every 3-4 days.

[0134] The negative control cells were cultured without differentiation factors, and the osteoblasts and a culture thereof were dispensed, and then collected on day 7, 14, or 21.

[0135] The cells were lysed using RIPA buffer, and then the expression of runt-related transcription factor 2 (RUNX2) was analyzed. Also for analysis of OCN secretion, the culture was collected, and normalized by cellular DNA.

[0136] Proteins were analyzed using SDS-PAGE on 12.5% polyvinylidene-Tris gels, and transferred on polyvinylidene difluoride (PVDF) membrane through electrophoresis.

[0137] The membrane was blocked using non-fat milk, and examined using anti-RUNX2 (D1H7) rabbit monoclonal antibody (#8486, Cell Signaling Technology, MA, USA) or anti-OCN (FL-95) antibody (# sc-30045, Santa Cruz Biotechnology, TX, USA). Anti-actin antibody (# M177-3, MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., Nagoya, Aichi, Japan) was used as an internal reference.

[0138] Protein bands on the blot were visualized using an enhanced chemiluminescent detection kit (# EBP-1073, PicoEPD Western Reagent, ELPIS-BIOTECH, Daejeon, Korea).

[0139] 7. Measurement of ALP Activity

[0140] ALP activity was assessed using Quantichrom ALP Assay Kit (Bioassay Systems, Hayward, Calif., USA) according to manufacturer's instructions.

[0141] Briefly, 310.sup.3 MC3T3-E1 cells were dispensed in a 96-well plate together with 10 L of complete -MEM containing ascorbic acid (50 g/mL) and 10 mM -glycerophosphate per well. Here, osmundacetone (10 M and 50 M) was added or not added, and the medium was exchanged every 3-4 days.

[0142] On day 14 of the incubation, the colorimetric change due to ALP activity in the cell fraction was measured using a spectrophotometer plate reader (Molecular Devices, Sunnyvale, Calif., USA) at 405 nm (Kim et al., 2016). The % activation of ALP activity was shown by comparing ALP activity of cells treated with an experimental compound and ALP activity of the non-treated control cells.

[0143] 8. TRAP Assay (Measurement of Osteoclast Proliferation and Differentiation Inhibitory Activity)

[0144] 1) Culture of Bone Marrow Cells

[0145] The tibia and femur of 6-8 week old male C57BL/6 mice were aseptically resected, and bone marrow cells were aseptically collected using a syringe (21 G, Korea Green Cross). The bone marrow cells were floated in 500 L of -MEM medium (Gibco BRL Co.) containing sodium bicarbonate (2.0 g/L), streptomycin (100 mg/L), and penicillin (100,000 unit/mL), dispensed in a 48-well plate, and assayed in triplicate. Mononuclear cells as precursor cells of osteoclasts were treated with RANKL and M-CSF as differentiation promoting factors, and thus differentiated into osteoclasts within 5-7 days.

[0146] 2) Measurement of Inhibition of Osteoclast Differentiation

[0147] 2-1) Sample preparation: The hot-water extract, EA extract, or fraction of Osmunda japonica were prepared by the same methods as in section 1 of <Example 1>. The EA-2 extract was dissolved in a minimum amount of DMSO, and then diluted with water to 70% of the original volume of the hot-water extract assuming that the yield of EA-2 extraction was approximately 70%. 2-2) Sample administration: The sample was continuously administered to a medium at 1:20 (v/v; 25 L of the sample per 500 L of the medium) from day 1 of incubation of bone marrow cells, while the medium was exchanged every 2-3 days. 2-3) Measurement of osteoclast differentiation Osteoclasts were defined by TRAP-positive multinucleated cells stained with TRAP. As for TRAP stain solution, 5 mg of naphthol AS-MS phosphate (Sigma N-4875) as a base and 25 mg of Fast Red Violet LB salt as a color developing reagent were dissolved in about 0.5 mL of N,N-dimethylformamide, and then mixed with 0.1N NaHCO.sub.3 buffer solution (50 mL) containing 50 mM tartaric acid. The reaction reagent was stored in a refrigerator before use.

[0148] After bone marrow cells were incubated in a medium containing differentiation promoting factors for 7 days, the medium was removed, and the cells were washed with PBS, and then immobilized with PBS containing 10% formalin for 2-5 minutes. Thereafter, the cells were immobilized with a 1:1 mixture solution of ethanol and acetone, followed by drying. The immobilized cells were treated with the TRAP stain solution for 15 minutes, and washed with PBS, and then the degree of cell staining was observed by a microscope.

[0149] In the microscope field of view, cells having two or more nuclei in the TRAP-positive cells were determined to be osteoclasts, and the number of cells was measured. The osteoclast differentiation inhibitory effect of the Osmunda japonica extract was calculated by IC.sub.50 as the 50% inhibitory concentration compared with the control group.

[0150] 9. TRAP Assay (Determination of Osteoclast Proliferation and Differentiation Inhibitory Activity IC.sub.50)

[0151] 1) Measurement of Osteoclast Differentiation

[0152] 1-1) Sample preparation: Osmundacetone was purchased from the Alfa Aesar, Thermo Fisher Scientific, and a minimal amount thereof was dissolved in dimethylsulfoxide (DMSA). Fosamax was purchased from Cayman (Ann Arbor, Mich., USA), and a minimal amount thereof was dissolved in sterile distilled water.

[0153] 1-2) Sample administration: Osmundacetone and Fosamax were continuously administered to a medium at 1:20 (v/v; 25 L of the sample per 500 L of the medium) from day 1 of incubation of bone marrow cells such that the final concentrations were 1, 4, 7, and 10 M, respectively, while the medium was exchanged every 2-3 days.

[0154] 1-3) Measurement of osteoclast differentiation: The measurement was carried out by the same method as in section 8 in <Example 1>.

[0155] In the microscope field of view, cells having two or more nuclei in the TRAP-positive cells were determined to be osteoclasts, and the number of cells was measured. The osteoclast differentiation inhibitory effect of osmundacetone was calculated by IC.sub.50 as the 50% inhibitory concentration compared with the control group.

[0156] 10. Investigation of Cytotoxicity

[0157] The compound isolated from Osmunda japonica, prepared in section 1 of <Example 1>, was investigated for cytotoxicity using MTT assay.

[0158] MTT assay was as follows.

[0159] Cells were incubated at 110.sup.3 cells/well in a 96-well plate containing DMEM with 10% fetal bovine serum (FBS) at 5% CO.sub.2 and 37 C., and then osmundacetone was added to the cell medium, followed by incubation for 24 hours. Thereafter, 100 L of MTT (0.5 mg/ml PBS) was administered, followed by incubation for 2 hours. Thereafter, the medium was removed from each well, and 100 L of DMSO was added. After incubation for 10 minutes, the absorbance was measured using a microplate reader (SPCTRA MAX 340PC, Molecular Devices, USA) at 570 nm. The absorbance is an indicator showing the number of living cells, and calculated by the following equation. The reproducibility thereof was validated by three experiments.

Cell proliferation (%)=OD.sub.550(sample)/OD.sub.550(control)

<Example 2> Results

[0160] 1. Confirmation of Osteoclast Proliferation and Differentiation Inhibitory Activity

[0161] The extract, fraction, and isolated compound of Osmunda japonica, prepared in <Example 1>, were investigated for osteoclast proliferation and differentiation inhibitory activity using the tartrate-resistant acid phosphatase (TRAP) assay, which is an osteoclast-specific staining method.

[0162] As can be confirmed from FIG. 2, giant osteoclasts were normally formed in the bone marrow cells treated with DMSO, like in the positive control group (a group with only differentiation promoting factors added to a culture medium without an Osmunda japonica extract). In contrast, the formation of giant osteoclasts as multinucleated cells was significantly inhibited in the group treated with the same volume of the water extract, EA extract, EA-2 fraction, and osmundacetone (10M) of Osmunda japonica similar to the negative control group (a group with neither differentiation promoting factors nor Osmunda japonica extract and added to a culture medium), and also, in addition to the results like in the negative control group, the proliferation of osteoclast precursor cells was significantly inhibited, leading to great inhibitory effects on both differentiation and proliferation of osteoclasts. Especially, when the cells were treated with 10 M osmundacetone, the differentiation of bone marrow mononuclear cells into multinucleated cells, osteoclasts, through proliferation and fusion was 95% or more, almost complexly inhibited. The group treated with 1 M osmundacetone showed the formation of giant osteoclasts, the number of which was smaller compared with the positive control group. The group treated with 1 M Fosamax also showed the formation of giant osteoclasts, the number of which was smaller compared with the positive control group, and showed an osteoclast density similar to that in the group treated with 1 M osmundacetone.

[0163] Also, the osteoclast proliferation and differentiation inhibitory activity IC.sub.50 was obtained. As can be confirmed from FIG. 3, the differentiation of bone marrow mononuclear cells into multinucleated cells, osteoclasts, through proliferation and fusion was almost completely inhibited when the cells were treated with 10 M osmundacetone (Alfa Aesar, Thermo Fisher Scientific), and was about 3-40% inhibited when the cells were treated with 7 M osmundacetone (IC.sub.50=8 M). The known osteoporosis medicine, Fosamax (alendronate), which was used as a positive control, had an IC.sub.50 value of 4 M (FIG. 4A).

[0164] In order to investigate whether or not osmundacetone inhibited the bone resorption function of completely differentiated mature osteoclasts, the bone resorption assay using mature osteoclasts was carried out in the presence of osmundacetone at 8 M, the IC.sub.50 concentration.

[0165] Osmundacetone inhibited the bone resorption function of mature osteoclasts up to 58.713% that of non-treated osteoclasts at the IC.sub.50 concentration on the basis of the method in section 5 of <Example 1>.

[0166] 2. Confirmation of Effects of Osmundacetone on Activation of Osteoblasts and Inhibition of Osteoclast Differentiation

[0167] In order to investigate whether or not osmundacetone has ability to inhibit osteoclast differentiation and activate osteoblast differentiation simultaneously, bone marrow mononuclear cells collected from C57BL/6 mice and MC3T3-E1 cells as osteoblast precursor cells were co-incubated in a 48-well plate at cell concentrations of 110.sup.5 bone marrow cells and 310.sup.3 MC3T3-E1 cells per well. The bone marrow monocyte/macrophage lineage cells in the co-incubated bone marrow cells and osteoblast precursor cells differentiated into mature multinuclear osteoclasts in the presence of M-CSF and RANKL within 6-7 days.

[0168] The administration of 10 M osmundacetone completely inhibited the proliferation and differentiation of osteoclasts (FIG. 4B, Os). Whereas the osteoblasts continuously proliferate and differentiate. The % activation values of ALP activity on days 7, 14, and 21 of co-incubation when 10 M osmundacetone was administered were 104%, 111%, and 95%, respectively, compared with when osmundacetone was not administered, and these results were not greatly different from the ALP activation of osteoblasts incubated alone after the administration of 10 M osmundacetone.

[0169] Osmundacetone showed similar inhibitory activities on osteoclast differentiation in the presence and absence of osteoblasts. On day 6 after the administration of 10 M osmundacetone, mature osteoclasts completely disappeared while osteoblasts continuously proliferated. Therefore, as shown from the co-incubation of preosteoclasts and preosteoblasts, osmundacetone did not show inhibitory activity on the activation and proliferation of co-existing osteoblasts.

[0170] On the basis of the above results, it was confirmed that osmundacetone has ability to inhibit osteoclast differentiation and activate osteoblast differentiation simultaneously.

[0171] In contrast, in the absence of osmundacetone, both osteoclasts and osteoblasts continuously proliferated and differentiated in the presence of osteoclast and osteoblast differentiation factors 7 days after the co-incubation of preosteoclasts and preosteoblasts (FIG. 4B, Positive control group).

[0172] However, the size of differentiated osteoclasts were somewhat small when compared with the size of osteoclasts grown in the absence of osteoblasts, and the reason may be due to cell density increased due to co-existence of osteoclasts and osteoblasts.

[0173] The cells grown in the incubation of the negative control group were most likely to configure macrophage/mononuclear cell lineage cells since only M-CSF was used as a differentiation factor (FIG. 4B, the negative control group).

[0174] Overall, these results indicated that osmundacetone has ability to inhibit osteoclast differentiation and activate osteoblast differentiation independently and simultaneously.

[0175] 3. Confirmation of Increasing Effects of Osmundacetone on ALP and OCN Production by Osteoblasts

[0176] In order to assess the ability of osmundacetone to induce bone formation, it was investigated whether or not osmundacetone increased the activity of alkaline phosphatase (ALP), which is a marker for initial/intermediate steps of osteoblast differentiation.

TABLE-US-00002 TABLE 2 % Activation of ALP activity of osmundacetone in MC3T3-E1 % Activation of ALP activity Compounds 10 M 50 M Osmundacetone 115 9.4 279 61* Parathyroid hormone-related 138 19 peptide (1 M)

[0177] The above values are expressed by means.d. of three independent experiments. * represents P<0.05. The parathyroid hormone-related peptide was used as a positive control.

[0178] As shown in Table 1 above, the ATP production in the osteoblast-like MC3T3-E1 cells treated with osmundacetone was significantly increased compared with ALP production in non-treated control cells.

[0179] Similar to the results shown in the previously known literature (Lyu et al., 2008), 50 M osmundacetone stimulated ALP production by 27961% (P<0.05).

[0180] In addition, osmundacetone showed a tendency to increase ALP production by MC3T3-E1 cells regardless of the inhibition of differentiation of co-existing osteoclasts. When osteoblast and osteoclast precursors were co-incubated with 10 M osmundacetone and osteoclast and osteoblast differentiation factors, mature osteoclasts completely disappeared on day 6 (FIG. 4B, Os).

[0181] Thereafter, the cells treated with osmundacetone were continuously grown in the presence of osteoblast differentiation factors.

[0182] The % activation values of ALP activity of the cells treated with osmundacetone, on days 7, 14, and 21 after the co-incubation of osteoblast and osteoclast precursor cells, were 104%, 111%, and 95%, respectively, compared with the non-treated control cells, and these results were almost similar to the % activation value obtained in the absence of co-existing osteoclasts (Table 1).

[0183] Therefore, the results showed that osmundacetone promoted osteoblast differentiation and, simultaneously, maintained ability to inhibit osteoclast differentiation.

[0184] It was reported that OCN, which is a main noncollagenous matrix protein, showed the most increased expression only at or near the time of mineralization, that is, about 21 days after the induction of MC3T3-E1 cell differentiation (Young et al., 1992).

[0185] In order to investigate the effect of osmundacetone on OCN production by osteoclasts, MC3T3-E1 cells were incubated in the presence of 50 M osmundacetone.

[0186] According to western blot assay, the levels of the OCN production by osteoblasts days 14 and 21 after the administration of osmundacetone were increased by 2.9 times and 1.2 times compared with the non-treated positive control cells, respectively (FIG. 5A, Os).

[0187] These results confirmed that osmundacetone initially induced a remarkable increase in OCN production by osteoblasts.

[0188] Even in the co-incubation of preosteoblasts and preosteoclasts, osmundacetone maintained ability to initially increase OCN production and inhibit osteoclast differentiation (FIG. 5B, Os).

[0189] The positive control osteoblasts grown in the presence of osteoblast and osteoclast differentiation factors in the environment of co-incubation without the administration of osmundacetone initially induced and increased the OCN production to a similar level to the osteoblasts in the environment of co-incubation with osmundacetone treatment, from day 7 to day 21 after cell dispensing (FIG. 5B, P.C). The reason is thought that the increase of OCN production was initially induced by the interaction with osteoclasts.

[0190] Interestingly, the negative control preosteoblasts grown without osteoblast differentiation factors, compared with osteoblasts grown without osteoclasts, showed a significant increase in OCN production to a level in the positive control cells in the presence of osteoclasts on days 14 and 21.

[0191] These results suggested that in the absence of osteoblast differentiation factors, the interaction with bone marrow mononuclear cells or mature osteoclasts may contribute to an increase in OCN production in osteoblasts.

[0192] Therefore, it was confirmed that osmundacetone inhibited osteoclast differentiation and maintained the ability to increase ALP and OCN expression in osteoblasts.

[0193] 4. RUNX2 Expression Increasing Effect of Osmundacetone

[0194] Then, it was investigated whether or not osmundacetone increased the expression of RUNX2, which is an initial differentiation marker of osteoblasts. It is known that RUNX2 is a transcriptional factor to increase bone formation by stimulating the transcription of ALP and OCN in osteoblasts (Phimphlai et al., 2006).

[0195] The expression levels of RUNX2 in MC3T3-E1 cells administered with osmundacetone on 7, 14, and 21 after the administration were increased by 1.1 times, 1.1 times, and 2.1 times compared with the non-treated positive control group (FIG. 6).

[0196] The RUNX2 expression in osteoblasts treated with osmundacetone was not significantly reduced until day 21 after cell dispensing, whereas the RUNX2 expression in non-treated osteoblasts on day 21 was reduced to almost half of the maximum expression level on day 14.

[0197] These results confirmed that osmundacetone had ability to extend the expression period of RUNX2 in osteoblasts.

[0198] 5. Cancer Cell-Specific Cytotoxicity of Osmundacetone

[0199] Osmundacetone showed specific toxicity with a higher selectivity index for cancer cells than non-cancer cells.

[0200] Compounds having an IC.sub.50 of less than 100 M may be considered to be active in cell death/anti-proliferative activity (Boyd, 2003).

[0201] Therefore, referring to Table 3 below, osmundacetone did not show cell cytotoxicity on normal cell lines.

TABLE-US-00003 TABLE 3 LD.sub.50 (M) Based on MIT Assay Normal cell Cancer cell Compounds GS 49 62 116 3 Cak-1 1060 10 $C-1 P3 Osmundacetone >5,000 >5,000 >5,000 >5,000 1.10 >5,000 >5,000 indicates data missing or illegible when filed

[0202] The above values are expressed by averagestandard deviation of three independent experiments. Fosamax was used as a reference compound.

[0203] The cells used in the present experiments were as follows.

[0204] HaCaT, human epidermal keratinocytes; ADMSC, human Adipose-derived mesenchymal stem cells (CEFO, Korea); RAW264.7, mouse macrophage cell line (preosteoclasts); NIH3T3, mouse embryo fibroblasts; AGS, human stomach adenocarcinoma; A549, human lung carcinoma; HepG2, human liver hepatoblastoma; HCT116, human colon carcinoma; PC3, human prostate adenocarcinoma; Caki-1, human kidney carcinoma; T24, human bladder carcinoma; HT1080, human fibrosarcoma; B16F10, mouse melanoma; TC-1 P3, HPV-16 E7-expressing mouse lung epithelial cells (-MHC class I)

[0205] Especially, according to MTT assay, osmundacetone showed IC.sub.50 values of 2,760220, 3510110, and >5,000 M for ADMSC human Adipose-derived mesenchymal stem cells, HaCaT human epidermal keratinocytes, and NIH3T3 mouse embryo fibroblasts, respectively, indicating slight toxicity.

[0206] Meanwhile, osmundacetone showed an LD.sub.50 of 50798 M for RAW264.7 mouse macrophage cell line, indicating relatively significant cytotoxicity. Therefore, these results suggested that osmundacetone may suppress the viability of some phagocytes at high concentrations.

[0207] Interestingly, according to MTT assay, osmundacetone showed low LD.sub.50 of 59.96.1, 65.58.7, and 75.89.2 M for cancer cell lines including AGS human stomach adenocarcinoma, PC3 human prostate adenocarcinoma, and B16F10 mouse melanoma, respectively, indicating moderate cytotoxicity activity.

[0208] Especially, the cancer selectivity index in cell cytotoxicity of osmundacetone was 45-60, very high in killing human cancer cell lines (AGS, PC3) compared with human normal cell lines (HaCaT, ADMSC).

[0209] In conclusion, the IC.sub.50 of osmundacetone for inhibition of osteoclast inhibition was 8 M, the concentration of osmundacetone to activate osteoblasts by 280% was 50 M, and the LD.sub.50 value of osmundacetone for several cancer cell lines was 50-70 M, whereas the LD.sub.50 of osmundacetone for normal cell lines was in a range of 2,500-5,000 M, and thus on the basis of these results, it was confirmed that osmundacetone had great safety when used as a medicine.

Application Example 1

[0210] Osteoporosis

[0211] Bone is maintained through a balanced bone remodeling cycle between osteoclasts that metabolically resorb bones and osteoblasts that form bones. However, when osteoclasts are extremely activated due to the destruction of the balance between osteoclasts and osteoblasts, the balance between bone resorption and formation is destroyed, and thus the amount of bone uptake is greater than the amount of bone formation, causing osteoporosis (Kim J H and Kim N, 2016; Shiozawa Y et al., 2011).

[0212] Therefore, osmundacetone of the present invention simultaneously shows an effect of inhibiting the proliferation and differentiation of osteoclasts and an effect of activating osteoblasts, and thus can exhibit a preventive or therapeutic effect on osteoporosis.

Application Example 2

[0213] Rheumatoid Arthritis

[0214] Rheumatoid arthritis is an autoimmune disease, and autoimmune antibodies promote osteoclast differentiation. The resultant excessive bone resorption worsens rheumatoid arthritis (Takayanagi H, 2007). The mechanism thereof is as follows. NFAT transcription factors (NFATc1/c2/c3/c4), which are key transcription factors related to osteoclast differentiation, are basically activated by calcium/calmodulin signaling (Takayanagi H et al., 2002). For complete activation, tyrosine-based activation motif (ITAM)-bearing molecules, such as the immunoregulatory protein DNAX-activating protein 12 (DAP12) and the immune antibody Fc receptor common chain (FcR), stimulates calcium signaling in immune cells (Pitcher L A and van Oers N S, 2003). Also, DAP12 and FcR activate NFATc1 through calcium signaling in osteoclasts. Therefore, immunoglobulin-like receptors involved in DAP12 and FcR play a key role in the differentiation of osteoclasts (Koga T et al., 2004; Mocsai A et al., 2004). That is, FcR interacts with osteoclast-associated receptor (OSCAR) and paired immunoglobulin-like receptor (PIR-A) in osteoclasts. The phosphorylation of ITAM activates phospholipase Cy (PLC), which is advantageous in the intracellular calcium, which activates calcineurin, which is calmodulin-dependent phosphatase. Calcineurin directly dephosphorylates serine of NFATc1, resulting in translocation into the nucleus, and activates NFATc1. Resultingly, the immune antibodies promote osteoclast differentiation, and excessive bone resorption by osteoclasts worsens rheumatoid arthritis. Ultimately, in rheumatoid arthritis patients, the inhibition of osteoclast differentiation cannot correct the abnormality of the autoimmune mechanism per se, but can treat skeletal symptoms, such as arthritis and pain resulting therefrom.

[0215] Therefore, osmundacetone of the present invention simultaneously shows an effect of inhibiting the proliferation and differentiation of osteoclasts, and thus can exhibit a preventive or therapeutic effect on rheumatoid arthritis.

Application Example 3

[0216] Paget's Disease (Osteitis deformans)

[0217] Paget's disease (Osteitis deformans) is also caused by abnormal bone resorption of osteoclasts (Singer F R, 2016). Therefore, abnormal osteogenesis of osteoblasts progresses, and this process is repeated, resulting in bone malformation, causing pains, headache, hearing loss, or the like, resulting therefrom. Paget's disease is frequently caused in arms, legs, pelvis, spine, and skull. The newly formed bone is weak, and thus the frequency of fracture is high. Hypercalcemia, heart attack, and hemiparesis may be caused (Ralstone S H, 2016). The cause of the disease is unknown, but genetic susceptibility and childhood virus infection are suspected to be the cause. The medication treatment is helpful in inhibiting the progression of the disease. Fosamax, an osteoclast differentiation inhibitor, and calcitonin, which regulates bone metabolism, are currently the most commonly used medicines. However, Fosamax is restricted in long-term use in some patients due to side effects thereof. Acetaminophen (Tylenol) or nonsteroidal anti-inflammatory drugs (NSAIDs) are used for severe pains.

[0218] Therefore, osmundacetone of the present invention shows an effect of inhibiting the proliferation and differentiation of osteoclasts, and thus can exhibit a preventive or therapeutic effect on Paget's disease.

Application Example 4

[0219] Bone Metastatic Cancer

[0220] Osteoclasts also promote bone metastasis of solid tumor. Bone is the most frequent site of cancer metastasis. The metastasis of cancer to bone causes severe pains and bone fractures, thereby significantly reducing the possibility of complete cure (Weilbaecher K N et al., 2011). Cancer cells spread throughout the body are found in proliferation sites of blood stem cells in the bone marrow (Shiozawa Y et al., 2013). Cancer cells significantly promote the differentiation of osteoclasts from bone marrow cells, thereby promoting bone metastasis, cancer growth, and bone destruction. Therefore, osteoclasts play a key role in the bone metastasis of cancer, and the inhibition of osteoclast differentiation reduces bone metastasis. Many solid cancer metastases correspond to bone metastasis, and blood stem cells are driven and grown based on blood stem cell proliferation sites, and then again comes into the blood, and metastasized to a different site. In prostate cancer, bone metastasis occurs most frequently, and such bone metastasis worsens cancer to make the cure of cancer difficult, resulting in a major cause of death. The direct main target of human prostate cancer cells is also a proliferation site of blood stem cells, and used as a key base of metastatic cancer (Shiozawa Y et al., 2011). In addition, osteoclasts promote angiogenesis in prostate cancer tissues, thereby promoting cancer growth (Bruni-Cardoso A et al., 2010). Breast cancer cells also promote osteoclast differentiation, and thus osteoclasts promote cancer recurrence through bone metastasis in breast cancer patients undergoing mastectomy (Danilin S et al., 2012; Lu X et al., 2011).

[0221] Bone-targeting therapeutic agents to prevent bone metastatic cancer are currently being used in clinical practice. Osteoclasts are one of the key mechanisms of bone metastasis of cancer, and thus become a major target of development of new anti-cancer drugs. Zoledronic acid is currently the only bisphosphonate-based drug, approved by the US FDA, for the purpose of inhibiting osteoclast differentiation (El-Amm J et al., 2013). Zoledronic acid preserves bones and increases survival rates. Zoledronic acid significantly reduced bone metastasis in high risk nonmetastatic prostate cancer (Wirth M et al., 2014). The co-administration of zoledronic acid with parathyroid hormone that activates osteoblasts further reduced bone metastasis (Schneider A et al., 2005). It was again verified that denosumab, a monoclonal antibody to RANKL, a signaling substance for osteoclast differentiation, also inhibited bone metastasis of prostate cancer, and thus the osteoclast inhibition is important for inhibiting bone metastasis of cancer (Smith M R et al., 2012). The administration of zoledronic acid inhibited osteoclast differentiation, thereby significantly inhibiting bone metastasis, even in patients with multiple myeloma (Zhuang J et al., 2012). That is, if an osteoclast inhibitor having few side effects and low cost is developed, such osteoclast inhibitor can be administered for a long time to inhibit metastasis in cancer patients.

[0222] Therefore, osmundacetone of the present invention simultaneously shows an effect of inhibiting the proliferation and differentiation of osteoclasts and an effect of activating osteoblasts, and thus can exhibit a preventive or therapeutic effect on bone metastatic cancer.

REFERENCES

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INDUSTRIAL APPLICABILITY

[0265] The compositions according to the present invention show strong inhibitory effects on osteoclast proliferation and differentiation and, simultaneously, activate osteoblasts, and thus can be favorably used to develop a safe and effective osteoporosis medicine or a safe and effective food for alleviating osteoporosis.