KR20210134134A - A pharmaceutical composition for preventing osteoclast differentiation comprising a physiologically active substance derived from an Echinacea extract - Google Patents

Abstract

According to the present invention, it is shown that a specific physiologically active substance derived from Echinacea has an excellent effect of inhibiting osteoclast differentiation. The present invention relates to a pharmaceutical composition for inhibiting osteoclast differentiation, including the physiologically active substance as an active ingredient, and the composition can be used as medicine or health-aid food. The present invention provides a pharmaceutical composition for inhibiting osteoclast differentiation, including Echinalkamide compound represented by chemical formula 1 and isolated from an Echinacea extract, as an active ingredient.

Description

A pharmaceutical composition for inhibiting osteoclast differentiation comprising a physiologically active substance derived from an Echinacea extract

The present invention relates to a pharmaceutical composition for inhibiting osteoclast differentiation, and more particularly, to a pharmaceutical composition for inhibiting osteoclast differentiation using an echinacea extract.

Bone is a calcified connective tissue associated with muscle, and the surface is thick and hard calcified tissue, providing basic and diverse functions such as protection of important organs and provision of a microenvironment necessary for hematopoiesis. It is also one of the important parts of the human body that stores essential minerals such as calcium, phosphorus, and magnesium in the body.

Bone metabolism consists of the action of osteoblasts responsible for bone formation and osteoclasts responsible for bone resorption, and the functions of these cells are balanced to maintain homeostasis. Therefore, a balance between them is important in bone metabolism. Bone tissue is an active metabolic tissue that is continuously remodeled, and the aged bone is dissolved by osteoclasts, and the bone matrix constituting the bone is removed by attaching to the surface of the bone and secreting an oxidase enzyme. On the other hand, in the formation of new bone, osteoblasts secrete calcium and phosphorus to form a skeleton. Bone resorption and bone formation by maintaining the homeostasis of these cells in vivo are important for maintaining the normal structure of the musculoskeletal system.

Osteoporosis, which is a representative example of a bone metabolic disease caused by an increase in osteoclast activity or an early activation of mature osteoclasts compared to osteoblast activity, refers to a symptom in which total bone mass decreases. In addition, breast cancer and prostate cancer are caused by bacteria that cause bone metastasis, primary bone-generated tumors (eg, multiple myeloma), rheumatoid or degenerative arthritis, and periodontal disease. There are periodontal disease in which the destruction of alveolar bone occurs, and Paget's disease caused by various genetic predispositions. These bone metabolic diseases are caused by imbalance in the interaction between osteoclasts and osteoblasts by factors such as hormones and physical stress in vivo. In particular, bone diseases associated with aging or osteoporosis caused by menopause in women are mainly caused by increased bone resorption activity of osteoclasts rather than bone formation.

Osteoclasts are cells derived from monocytes of hematopoietic stem cells, and when RAW2647 monocytes from mice bind to RANKL (receptor activator of nuclear factor KB (RANK) ligand), they mature into multinucleated osteoclasts and resorb bone. (bone resorption) occurs.

RANKL (receptor activator of nuclear factor κB ligand) produced in osteoblasts or activated immune cells promotes the formation and activity of osteoclasts by binding to receptors located in osteoclasts and progenitor cells (RANK). In addition, osteoblasts produce RANKL and its decoy receptor OPG (osteoprotegerin).

Osteoprotegerin (OPG) is an in vivo antagonist of RANKL. That is, the formation and activity of osteoclasts is regulated by the balance of RANKL and OPG. However, when stimulation of bone resorption such as estrogen deficiency or hyperparathyroidism occurs, the balance between OPG and RANKL is disrupted, and osteoclasts are activated to promote bone resorption. Therefore, blocking the signaling pathway activated by RANKL is recognized as one of the therapeutic approaches for the treatment of osteoporosis as well as bone diseases.

On the other hand, Echinacea (Echinacea) is a perennial herb plant of the Asteraceae family, native to North America, and grows to about 80 to 120 cm in size. Leaves are alternate phyllotaxis, hairless, and the tip is lanceolate and long oval. The flowers bloom from July to October as head and snow flowers. The capsicum is purplish, and the snow cape blooms horizontally, but droops downward when it grows to more than 10 cm.

In addition, the scientific name of Echinacea is Echinacea pupurea , and Echinacea is one of the herbal plants popular for medicinal and landscaping with large and colorful flowers.

In relation to the use of these, echinacea, Patent Publication No. 2002-7011626 discloses the anti-aging effect of the ecanacea extract, etc., and the osteoclast differentiation inhibitory effect is not clearly known, and also, the echinacea It is not clearly known which of the various physiologically active substances has an effect on the inhibition of osteoclast differentiation in particular.

Therefore, the present invention has been devised to solve the above problem, revealing a specific physiologically active substance derived from Echinacea that shows excellent efficacy in inhibiting osteoclast differentiation, and inhibiting osteoclast differentiation containing this physiologically active substance as an active ingredient As a pharmaceutical composition, it is intended to provide a composition that can be used as a pharmaceutical or health functional food.

In order to solve the above problems, the present invention provides a pharmaceutical composition for inhibiting osteoclast differentiation containing, as an active ingredient, an echinalkamide compound represented by the following Chemical Formula 1 isolated from an echinacea extract.

[Formula 1]

According to the present invention, when preparing a pharmaceutical composition for inhibiting osteoclast differentiation using an echinacea extract by clearly suggesting that the echinacea-derived physiologically active substance, which exhibits excellent efficacy in inhibiting osteoclast differentiation, is an Echinalkamide compound, It has the effect of providing an efficient manufacturing standard by controlling the content of substances.

In addition, the composition according to the present invention is derived from an extract extracted from a natural product and has almost no side effects, and has an osteoclast differentiation inhibitory effect.

1 is a graph showing the results of measuring the cytotoxicity of echinalcamide;
2 is a graph showing the results of measuring the osteoclast differentiation inhibitory ability of echinalcamide;
3 is a photograph taken with an inverted microscope (Leica microsystems, Germany) to confirm the osteoclast differentiation inhibitory ability of echinalcamide;
Figure 4a is a photograph taken with a FluorChem E system image analyzer (Cell Biosciences, Santa Clara, CA) the result of measuring the protein expression of NFATc1 and c-Fos by echinalcamide;
Figure 4b is a graph showing the results of quantification of the protein expression of NFATc1 and c-Fos by echinalcamide with Image J;
Figure 4c is a graph showing the results of measuring the mRNA expression of NFATc1 and c-Fos by echinalcamide;
Figure 5a is a photograph taken with the FluorChem E system image analyzer (Cell Biosciences, Santa Clara, CA) the result of measuring the protein expression of MMP-9 and cathepsin K by echinalcamide,
Figure 5b is a graph showing the results of quantification of the protein expression of MMP-9 and cathepsin K by Image J by echinalcamide,
Figure 5c is a graph showing the results of measuring the mRNA expression of MMP-9 and cathepsin K by echinalcamide,
Figure 6a is a picture taken with a FluorChem E system image analyzer (Cell Biosciences, Santa Clara, CA) the result of measuring the protein expression of p-ERK, p-P38 and p-JNK by echinalcamide,
Figure 6b is a graph showing the results of quantification of the protein expression of p-ERK by Image J by echinalcamide,
Figure 6c is a graph showing the results of quantification of the protein expression of p-P38 by Image J by echinalcamide,
Figure 6d is a graph showing the results of quantification of the protein expression of p-JNK by Image J by echinalcamide,
Figure 7a is a photograph taken with an inverted microscope (Leica microsystems, Germany) to confirm the bone resorption ability of echinalcamide;
Figure 7b is a graph showing the results of measuring the bone resorption capacity of echinalcamide.

Hereinafter, preferred embodiments of the present invention will be described in detail. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

The present inventors discovered a new compound derived from cultured Echinacea root, and filed a patent application on June 29, 2018 with the title of the invention "new compound derived from cultured Echinacea and its anti-inflammatory use", and in this patent, After separation of 11 compounds, some compounds (Compounds 1 to 3) showed anti-inflammatory activity through inhibition of NO and TNF-α production, but the possibility of inhibiting actual osteoclast formation and bone resorption could not be confirmed. , it was also difficult to predict which of the 11 isolated compounds would have such an effect. However, in the present invention, it was confirmed that the echinacea extract showed an excellent effect on inhibiting osteoclast formation and bone resorption by inhibiting osteoclast differentiation, and osteoclast differentiation among the physiologically active substances contained in the echinacea extract. As a result of repeated research to search for substances with specific activity for inhibition and improvement of bone diseases, it was confirmed that the effective substance exhibiting functionality in osteoporosis, improvement of bone disease and inhibition of osteoclast differentiation is Echinalkamide compound. came to the invention.

Accordingly, the present invention discloses a pharmaceutical composition for inhibiting osteoclast differentiation containing an echinalkamide compound represented by the following formula (1) isolated from an echinacea extract as an active ingredient.

[Formula 1]

The Echinacea extract can be prepared by appropriately adopting a method known in the art from Echinacea, and thus conditions such as extraction time, solvent, temperature, etc. can be appropriately adjusted by a person skilled in the art, preferably ethanol or ethyl It can be extracted using acetate.

The pharmaceutical composition according to the present invention can inhibit osteoclast formation and bone resorption. In addition, the pharmaceutical composition of the present invention may be applied to treat or prevent bone disease. Examples of bone diseases include, but are not limited to, arthritis, osteoporosis, rheumatoid arthritis, Paget's disease, scoliosis, fibrodysplasia, osteolytic metastasis, pycnodysotosis, and periodontal disease.

The pharmaceutical composition according to the present invention may be used in combination with a conventional osteoporosis treatment. In this case, due to the combined use of osteoporosis treatment with existing limitations, such as bisphosphonates, SERM (selective estrogen receptor modulator), calcitonin, estrogen, vitamin D complex, denosumam, and parathyroid hormone (PTH), not only efficacy but also side effects are reduced. is expected to be minimized. In addition, it is possible to present the possibility of developing a new therapeutic agent through stability evaluation.

The pharmaceutical composition for inhibiting osteoclast differentiation comprising an echinalkamide compound according to the present invention may be used in the form of a pharmaceutical or health functional food depending on the purpose.

When used as the pharmaceutical, it may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. In addition, the pharmaceutical composition can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, and can be used by formulating the extracts. Carriers, excipients and diluents that may be included in the composition comprising lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

When used as the health functional food, ingredients commonly used in health functional food may be included in addition to the active ingredient, for example, citric acid, oligosaccharide, taurine, fruit concentrate, etc. may be included when manufacturing a health functional beverage.

Hereinafter, the present invention will be described in detail by way of examples.

Preparation Example: Preparation of mouse BMM

Femurs and tibias were washed with alpha-minimum essential medium (α-MEM) supplemented with 10% FBS (fetal bovine serum), penicillin (100 U/mL) and streptomycin (100 mg/mL) to 5 week-old male C57BL6 mice. Mouse BMC (Bone Barrow Cells) was obtained from (Orient Bio, Gwangju). To obtain bone marrow-drived macrophages (BMM), BMCs were seeded on a culture dish in α-MEM supplemented with 10% FBS and macrophage colony-stimulating factor (M-CSF, 10 ng/mL), and cultured for 1 day. Non-adherent cells were transferred to a 10 cm Petri dish and cultured for 3 more days in the presence of M-CSF (30 ng/mL). After removal of non-adherent cells, adherent cells were used as osteoclast precursors, BMM.

Example 1: Preparation of echinacea extract

With reference to prior literature (Murphy HN, Kim YS, Park SY, Paek KY. Biotechnological production of caffeic acid derivatives from cell and organ cultures of Echinacea species. Appl Microbiol Biotechnol. 2014; 98: 7707-7717) commercially available) was added to Murashige & Skoog medium (MS 2.2 g/L, agar 8 g/L, pH 6.0) was obtained through culture in a large-scale bioreactor under dark conditions. To 50 g of the cultured Echinacea sample, 95% (v/v) ethyl acetate was added 10 times and extracted at 20° C. for 24 hours to prepare an Echinacea ethyl acetate extract.

Example 2: Separation of bioactive substances

The echinacea ethyl acetate extract (4.7 g) prepared in Example 1 was _{subjected to silica gel column chromatography with CH 2} Cl ₂ -MeOH to obtain 15 fractions (E1-E15). After dissolving the E6 fraction in MeOH (100%), four fractions (E6A-E6D) were obtained through column chromatography (Sephadex LH-20). After dissolving the obtained E6B in CH ₃ CN-H ₂ O (25:75), a peak eluted at 28.3 minutes was obtained through column chromatography (semi-preparative HPLC), and echinalkamide was separated and purified. . Echinalcamide was identified as a novel compound previously unknown as follows.

Echinalcamide was isolated as a brown syrup. _{The molecular formula was confirmed as C 22} H ₃₁ NO ₇ from HREIMS (m/z 444.1992 ([M + Na]+, calcd for 444.1998)) and ¹³ C NMR data. The IR spectrum showed typical absorption bands of hydroxyl and carbonyl groups at 3364 and 1646 cm ^{-1 , respectively. 1} H and ¹³ C NMR spectra show the characteristic anomeric proton δH 4.41 (1H, d, J, 80 Hz, H-1") and six glucosyl carbons (δC 100.9, 76.7, 76.6, 73.7, 70.2, 61.3). It was observed that the presence of glucose residues could be inferred.The arrangement of glucose residues was determined to be β based on the coupling constant of J = 8.0 Hz.H-3'/4' and C-1', C-2 'of the ¹ H and ¹³ C NMR spectrum with the HMBC correlation between _{[δ H 3.03 (2H, d} , J = 7.0 Hz, H-1'), 1.78 (1H, m, H-2 ') and 0.91 ( signals at _{δ C 46.4 (C-1 '} ), 28.3 (C-2'), 19.2 (C-3 ', 4')]; 6H, d, J = 7.0 Hz, H-3 ', 4') In addition, the _{1 H NMR data showed [δ H} 5.67 (1H, d, J = 11.5) in the Z and E configurations determined by the coupling constants of J = 11.5 Hz and 15.5 Hz, respectively. Hz, H-2), 6.41 (1H, dd, J= 11.5, 11.5 Hz, H-3)] and [δ _H 7.45 (1H, ddd, J= 15.5,.11.5, 1.0 Hz, H-4), 5.98 (1H, dt, J = 15.5, 6.5 Hz, H-5)], the signals of two pairs of olefinic protons were also observed, and in the ¹ H NMR spectrum, two methylene signals, δH2.39 (2H, m, H -6), 2.43 (2H, m, H-6) and one oxymethine signal, δH4.47 (2H, m, H-12), were observed in the ¹³ C NMR spectrum, which is ^{consistent with the 1} H NMR data. Signals are shown for two pairs of olefinic carbons, two methylenes and one oxymethylene.Also, in the ¹³ C NMR and HSQC spectra, the four quaternary carbons in [δC79.6, 64.5, 70.7, 71.7] are 2 Assign as a triple bond of a pair became ^{Interpretation of 1} H and ¹³ C NMR data showed that echinalcamide was very similar to that of undeca-2Z-4E-diene-8,10-diynoic acid isobutylamide, except for the presence of oxymethylene and glucose residues instead of methyl groups. It is proposed that echinalcamide is the glucoside of undeca-2Z-4E-diene-8,10-diynoic acid isobutylamide. This is supported by the HMBC correlation. The positions of the isobutyl and glucose moieties were determined as C-1 and C-12, respectively, by HMBC correlations between H-1' and C-1 and between H-1'' and C-12. Accordingly, echinalkamide was determined to have a structure as shown in Chemical Formula 1 below, which is a compound reported to be isolated and reported for the first time in nature, and was named “echinalkamide” in the present invention.

Echinalkamide. Brown syrup; 21.2 (c 0.03, MeOH); UV (MeOH)λ max 249.0 nm; IRmax3364,1646cm-1;1H NMR (500 MHz, CD3OD)δ5.67(1H,d,J = 11.5 Hz, H-2), 6.41 (1H, t, J = 11.5 Hz, H-3), 7.45 ( 1H, ddd, J = 15.5, 11.5, 10 Hz, H-4), 5.98 (1H, dt, J = 15.5, 6.5 Hz, H-5), 2.39 (2H, m, H-6), 2.43 (2H) , m, H-7), 4.47 (2H, s, H-12), 3.03 (2H, d, J = 7.0 Hz, H-1′), 1.78 (1H, m, H-2′), 0.91 ( 6H, d, J = 7.0 Hz, H-3′, 4′), 4.41 (1H, d, J = 8.0 Hz, H-1″), 3.1-3.3 (4H, m, H-2″, 3″) , 4″, 5″), 3.84 (2H, m, H-6″) ppm; 13CNMR (125MHz, CD3OD),δ167.6(C-1), 119.4(C-2), 140.2(C-3), 128.2(C-4), 139.3(C-5), 31.2(C-6) , 18.2(C-7), 79.6(C-8), 64.5(C-9), 70.7(C-10), 71.1(C-11), 55.7(C-12), 46.4(C-1′) , 28.3(C-2′), 19.2(C-3′,4′), 100.9(C-1″), 76.7(C-2″), 76.6(C-3″), 73.5(C-4″) ), 70.2 (C-5″), 61.3 (C-6″) ppm; ESIMS (positive mode) m/z: 444 [M+Na]+; HRESI-TOF-MS (positive mode) m/z: 444.1492 (calcd for C22H31NNaO 744.41998)

[Formula 1]

Experimental example

(1) Cytotoxicity measurement

In order to determine whether intracellular treatment of Echinalkamide and RANKL (Receptor Activator of Nuclear factor Kappa B Ligand) induces cytotoxicity, the BMM prepared in Preparation Example was added to a 96-well plate (1x10 ⁴ cells). / well), and after overnight incubation, M-CSF (30 ng/mL) and the test substance prepared in Example 2 were treated at a concentration of 1 μM to 50 μM, respectively. After 3 days of incubation, 50 μl XTT (sodium 3′-[1-(phenyl-aminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate and N-methyl Dibenzopyrazine methyl sulfate) reagent was added to each well, and then the cells were incubated for 4 hours. After incubation, the optical density of each well was measured at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, Calif.).

(2) osteoclast formation assay

In order to generate osteoclasts, the BMM prepared in Preparation Example was placed in a 96-well plate (1x10 ⁴ cells/well) in a complete medium containing M-CSF (30 ng/mL) and RANKL (100 ng/mL). After sowing, the test substances prepared in Example 2 were treated with concentrations of 0 μM, 1 μM, 5 μM, 10 μM and 30 μM, respectively, and cultured for 4 days. In order to generate osteoclasts from a co-culture of primary osteoblasts and BMCs, at the end of the culture period, the cells were fixed in 3.7% formalin for 10 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and then TRAP (Sigma -Aldrich). TRAP-positive multinucleated cells with more than 3 nuclei were counted as osteoclasts.

(3) Real-time reverse transcriptase PCR

BMM cells treated with RANKL and RANKL and Echinalkamide for 24, 48, 72 and 96 hours, respectively, were extracted with eszy-Blue ^TM Total RNA extraction kit (iNtRON Biotechnology, Korea) and mRNA was extracted, followed by TaqMan RNA-to - Mix ^{extract and primer using Ct TM} 1-Step Kit and repeat 40 intrinsic 60 times in StepOne machine at 48°C for 15 minutes, 95°C once for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute to measure the Ct (threshold cycle) value.

(4) Western blot analysis

After the BMM cells prepared in Preparation Example were treated with RANKL and RANKL and Echinalkamide, respectively, the culture medium of the pellet was completely removed with PBS (PHOSPHATE BUFFERED SALINE), and proteinase inhibitors were included. Lysis buffer (lysis buffer) was dissolved on ice for 30 minutes. After centrifuging the lysate at 13,000 rpm for 25 minutes, the supernatant was taken and the protein obtained was quantified and used. Protein and 5Хsample buffer were mixed and inactivated by heating at 95°C for 5 minutes, followed by electrophoresis on SDS polyacrylamide gel. The protein of the electrophoresed gel was moved to a nitrocellulose membrane, blocked with skim milk or BSA for 1 hour, and then washed. Dilute the primary antibody (c-FOS, NFATc1, MMP-9, Cathepsin K, phospho-ERK, phospho-JNK, phospho-P38) in the membrane, react at 4°C for 24 hours, wash with PBST, and then primary-specific The HRP conjugate secondary antibody was reacted at room temperature for 1 hour. After washing with PBST, light was emitted with ECL solution, and bands were photographed using a FluorChem E system image analyzer (Cell Biosciences, Santa Clara, CA). The increase or decrease of protein was quantified using Image J (Media Cybernetics, Silver Spring, Maryland, USA) program.

(5) Bone resorption analysis

In order to evaluate the bone resorption capacity resulting from the differentiation and activity of osteoclasts, artificial bone (Corning Life Sciences, Corning Inc, Corning, NY, USA) prepared by coating a plate with calcium-collagen was used. measured. The plate coated with calcium phosphate and collagen was washed with phenol-red-free MEM medium. After washing, undifferentiated osteoclast progenitor cells were aliquoted at 1Х10 ³ cells/well, and after 24 hours incubation, RANKL was treated with 100 ng/ml. Echinalkamide prepared in Example 2 was treated with RANKL-treated medium at concentrations of 1 μM, 10 μM and 30 μM, and then cultured for 7 days, and the medium was replaced once every 2 days. After incubation, cells were removed by treatment with 5% sodium hypochlorite, washed with deuterium depleted water (DDW), and bone resorption was observed with a Х400 inverted microscope (Leica microsystems, Germany) and photographs were taken. After imaging, the resorption site was quantified with Image J software (Media Cybernetics, Silver Spring, Maryland, USA).

(6) Statistical analysis

Experiments were performed at least 3 times, and data are expressed as mean±standard deviation (SD). All statistical analyzes were performed using the Statistical Package (SPSS; Korean version 14.0) of Social Sciences Software. Student's t-test was used to compare variables between the two groups, and P < 0.05 was considered statistically significant.

Osteoclasts are cells of the monocyte macrophage lineage uniquely responsible for bone destruction in bone tissue, and can be produced by differentiation from monocyte macrophage progenitors present in various tissues by specific cytokines such as RANKL.

Referring to FIG. 1 , it was determined that Echinalkamide had no toxicity up to a concentration of 30 μM.

2 and 3 , when TRAP staining is performed, in the control group not treated with RANKL, BMC cells proliferate while maintaining a spherical shape, and also show a negative reaction in TRAP staining, resulting in light brown or ocher color. In contrast, in the cells treated with RANKL, TRAP(+) multinucleated cells stained in dark dark brown to reddish brown are observed. In the RANKL-treated cell group used as a positive control, differentiation was induced into TRAP(+) multinucleated cells having three or more nuclei.

4A to 4C, the effect of echinalcamide on the expression of key transcription factors, such as c-Fos and NFATc1, can be seen to determine the molecular mechanism of the echinalcamide effect upon osteoclast formation. Expression of c-Fos and NFATc1 was upregulated in BMM by RANKL stimulation. Pretreatment with echinalcamide effectively inhibited RANKL-induced mRNA expression of c-Fos or NFATc1 at 24 hours or 48 hours, respectively, and also suppressed RANKL-induced c-Fos and NFATc1 protein expression.

5A to 5C , the RANKL-induced expression of MMP-9 and cathepsin K, two other markers of osteoclast formation, was also significantly reduced in the presence of echinalcamide.

6A to 6D , RANKL-induced early signaling including ERK, p38 and JNK to further investigate the molecular mechanisms supporting the inhibitory effect of Echinalkamide on RANKL-induced osteoclast formation. The effect of echinalcamide on the pathway is known. Phosphorylation of these signaling molecules was observed 15 min after RANKL treatment in BMM. Activation of ERK, p38 and JNK by RANKL was all significantly inhibited by echinalcamide.

7a and 7b, the pit area formed due to bone resorption after osteoclast formation by treatment with RANKL was large, and when treated with echinalcamide, bone resorption was inhibited at a concentration of 1 μM and small pits were observed. became When echinalcamide was treated at a concentration of 30 μM, pit formation was hardly observed.

As such, the pharmaceutical composition for inhibiting osteoclast differentiation comprising the echinalkamide compound according to the present invention, echinalkamide prevents RANKL-induced osteoclast formation and mature osteoclast bone resorption of BMM, In addition, since ekinalcamide is associated with downregulation of ERK, p38 and JNK, as well as c-Fos and NFATc1, and decreases the expression of MMP9 and cathepsin K, it is useful for the prevention or treatment of osteoclast-related bone diseases including osteoporosis. suggest that it can be used.

As above, preferred embodiments of the present invention have been described in detail with reference to the drawings. The description of the present invention is for illustrative purposes, and those of ordinary skill in the art to which the present invention pertains will understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention.

Therefore, the scope of the present invention is indicated by the claims to be described later rather than the above detailed description, and all changes or modifications derived from the meaning, scope, and equivalent concept of the claims are included in the scope of the present invention. should be interpreted

Claims (1)

A pharmaceutical composition for inhibiting osteoclast differentiation comprising an echinalkamide compound represented by the following formula (1) separated from the echinacea extract as an active ingredient.
[Formula 1]

Images