KR101765141B1 - Composition for prevention or treatment of bone disease containing skullcapflavone derivatives or pharmaceutically acceptable salts thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating bone diseases, comprising skullcapflavone derivatives or pharmaceutically acceptable salts thereof as an active ingredient. According to the present invention, it was confirmed that when BMM cells are treated with RANKL in the presence of the skullcapflavone derivatives, the differentiation of osteoclasts is significantly suppressed, and the expressions of the following members are suppressed: NFATc1 related to the differentiation of osteoclasts and target genes thereof; c-Fos which is known to be an upstream regulator of NFATc1; MAPKs and Src-AKT which are activated by RANKL; CREB-PGC-1 which is involved in mitochondrial biogenesis and target genes thereof; and caveolin-1 which promotes the differentiation of osteoclasts. Additionally, as the expressions of a redox-sensitive transcriptional factor, Nrf2 and target protein thereof which is an antioxidant enzyme, and FOXO1 and catalase are increased, the production of active oxygen species is decreased, bone resorption function of osteoclasts is reduced, and inflammation-induced bone damage is suppressed. Accordingly, the skullcapflavone derivatives of the present invention can be beneficially used in preventing or treating bone diseases.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis, which comprises, as an active ingredient, a scalarcaflavone derivative or a pharmaceutically acceptable salt thereof,

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis, which comprises a scalarcaflavone derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Osteoporosis, a typical metabolic bone disease, is a disease in which lime in the bone tissue is reduced and the density of the bone becomes thinner, thereby widening the bone marrow cavity. As the symptoms progress, the bone becomes weaker, It is easy to fracture even in impact.

Osteoporosis is characterized by a decrease in bone mass and an abnormality in microstructure. As aging progresses, the balance between the absorption of old bone and the formation of new bone is broken and new bone replacement (bone remodeling) is not smooth, , The risk of breakage or breakage increases. These bone masses are affected by various factors such as genetic factors, nutritional intake, hormonal changes, exercise and lifestyle differences, and are affected by age, lack of exercise, underweight, smoking, low calcium diet, menopause, ovariectomy It is a major cause of bone loss.

On the other hand, there is an individual difference, but the bone mass is usually the highest at 14-18 years old, and decreases about 1% per year at old age. Especially in women, bone reduction continues after 30 years of age, and bone turnover is rapidly progressed by hormonal changes in menopause. B-lymphocytes are produced in the bone marrow as a result of IL-7 (interleukin-7), and B-cell precursors (pre-B Cells accumulate, which increases the amount of IL-6, which increases the activity of osteoclasts, leading to a decrease in bone mass.

Thus, osteoporosis is a symptom that is inevitable for older people, especially postmenopausal women, and owing to the aging population in developed countries, interest in osteoporosis and its therapeutic agents is increasing gradually. In addition, there is a market of about $ 130 billion related to the treatment of bone disease worldwide, and since it is expected to increase further in the future, each research institute and pharmaceutical company in the world invests heavily in the development of bone disease treatments.

In Korea, the average life span is approaching 80 years, and the prevalence of osteoporosis is rapidly increasing. According to recent studies conducted by local residents, 4.5% of men and 19.8% . This suggests that osteoporosis is a more common health problem than diabetes or cardiovascular disease and that osteoporosis is a very important health problem when estimating the cost of suffering or treatment for patients receiving fractures.

Several substances have been developed to treat osteoporosis. Estrogen, the most commonly used drug for osteoporosis treatment, has not yet been proven its practical efficacy. It has the disadvantage of continuing to take it during its lifetime, and there is a side effect of increasing the risk of breast cancer or uterine cancer when administered over a long period. Alrendronate is also less effective, has less absorption in the digestive tract, and causes inflammation in the stomach and esophageal mucosa. Calcium preparations are known to have good efficacy with fewer side effects, but they are more preventive than treatments. In addition, vitamin D preparations such as calcitonin are known, but studies on efficacy and side effects have not yet been conducted. Therefore, there is a need for a new metabolic bone disease treatment agent having less side effects and excellent effects.

Osteoclasts are large, multicellular cells that destroy or absorb bone tissue that has become unnecessary in the process of bone growth of vertebrates, and are cells differentiated from osteoclast precursors. The osteoclast precursor cells differentiate into osteoclasts in the presence of M-CSF and RANKL and form multinucleated osteoclasts through fusion. The osteoclasts bind to the bone through αvβ3 integrin and the like to form an acidic environment and secrete various collagenase and protease to cause bone resorption. Inhibition of osteoclast Can be an effective method of treating bone diseases.

Meanwhile, skullcapflavone derivatives are flavonoids derived from Scutellaria baicalensis (gold), which are known to have anti-inflammatory and anticancer effects by folk remedies. They are antagonists of bradykinin and are involved in cell signaling (Yun-Choi et al., Archives of Pharmacology Research, v.16 no.4, 1993, 283-288). Korean Patent Laid-Open Publication No. 2013-0120849 also discloses the use of a scalarcaflavone derivative as a pharmaceutical material as a composition for preventing or treating asthma. However, the osteoclast differentiation control effect and the bone disease treatment use of the scalarcaflavone derivative of the present invention .

Accordingly, the present inventors have searched for a substance that inhibits the activity of osteoclasts in order to find a new substance for treating bone diseases, and found that the scalarcaflavone derivative inhibits osteoclast-induced bone resorption, Can be effectively used as a pharmaceutical composition for treating bone diseases, the present invention has been completed.

It is an object of the present invention to provide a pharmaceutical composition containing, as an active ingredient, a scalarcaflavone derivative or a pharmaceutically acceptable salt thereof, for the prevention or treatment of bone diseases.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating bone diseases, which comprises a scalarcaflavone derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food for improving osteoporosis, which comprises a scalarcaflavone derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition for preventing or treating bone diseases containing the scalarcaflavone derivative of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient inhibits important factors involved in osteoclast differentiation and thus has an effect of inhibiting bone resorption And can be useful for preclinical and clinical studies. In addition, since bone-related diseases caused by imbalance of osteoclasts can be treated, it can be widely used for the prevention or treatment of bone related diseases.

Brief Description of the Drawings Figure 1 shows the concentration and time-dependent effects of SF II, a composition of the present invention, on the differentiation of osteoclast precursor cells (BMMs) treated with RANKL:
A: TRAP staining of osteoclasts differentiated by RANKL in the presence of various concentrations of SF II;
B: Analysis of TRAP-stained multinuclear osteoclast numbers of A above;
C: Cytotoxicity analysis of concentrations of SF II (M: M-CSF / M + R: M-CSF and RANKL);
D: TRAP staining of osteoclasts differentiated by RANKL for various times in the presence of SF II;
E: Analysis of TRAP-stained multinuclear osteoclast numbers of D above; And
F: Cytotoxicity analysis of SF II over time (Veh: Vehicle).
Figure 2 shows the effect of SF II, a composition of the present invention, on the expression of NFATc1 and target genes of osteoclast precursor cells (BMMs) treated with RANKL:
A: Effect of concentration of SF II on the expression of NFATc1;
B: hourly effect of SF II on expression of NFATc1; And
C: Effect of SFII on mRNA expression of NFATc1 and target genes.
Figure 3 shows the effect of SF II, a composition of the present invention, on NF-κB activity and c-FOS expression in RANKL-treated osteoclast precursor cells (BMMs)
A: The effect of SF II on phosphorylation and degradation of IκBα;
B: Effect of SF II on expression of NF-κB target gene;
C: Effect of SF II on the expression of c-Fos; And
D: Effect of SF II on mRNA expression of c-Fos and NF-κB target genes.
Figure 4 shows the effect of SF II, a composition of the present invention, on the phosphorylation of MAPKs, Src-AKT and FOXO1 in RANKL-treated osteoclast precursor cells (BMMs)
A: Effect of SF II on phosphorylation of MAPKs;
B: Effect of SF II on phosphorylation of Src-AKT and FOXO1;
C: Effect of SF II on FOXO1 and catalase expression; And
D: Effect of SFII on mRNA expression of catalase.
Figure 5 shows the effect of SF II, a composition of the present invention, on the activity of CREB-PGC-1? In osteoclast precursor cells (BMMs) treated with RANKL:
A: Effect of SF II on mRNA expression of PGC-1? And target genes;
B: the effect of SF II on the expression of PGC-1?; And
C: Effect of SFII on phosphorylation of CREB.
FIG. 6 shows the effect of SF II, a composition of the present invention, on the production of Nrf2 and its target genes, caveolin-1, and active oxygen in RANKL-treated osteoclast precursor cells (BMMs)
A: Effect of SF II on mRNA expression of Nrf2 and target genes;
B: Effect of SF II on the expression of target proteins, including Nrf2 and antioxidant enzymes;
C: Effect of SF II on intracellular active oxygen accumulation; And
D: Effect of SFII on the expression of caveolin-1.
FIG. 7 shows the effects of exposure time of SF II, a composition of the present invention, on osteoclast differentiation and expression of NFATc1 and its target genes:
A: Effect of exposure time of SF II on osteoclast differentiation; And
B: Effect of exposure time point of SF II on mRNA expression of NFATc1 and target genes.
FIG. 8 is a diagram showing the effects of exposure time of SF II, the composition of the present invention, on the actin ring formation of osteoclast precursor cells (BMMs) treated with RANKL and the bone resorption in dentine discs:
A: A diagram showing the experimental design of the SF II at the time of exposure;
B: Effect of exposure time point of SF II on the formation of actin ring; And
C: Effect of exposure time of SFII on bone resorption.
Figure 9 shows the effect of SF II, a composition of the present invention, on bone damage caused by LPS-induced inflammation;
A: Photograph of a TRAP stained skull;
B: photographs of the skull sections stained with TRAP and hematoxilin; And
C: Analysis of the bone cavity and number of osteoclasts in B above.

Hereinafter, the present invention will be described in detail.

The skullcapflavone derivative containing the present invention as an active ingredient is a flavonoid derived from Scutellaria baicalensis (golden), and has a structure represented by the following formula (1):

(Wherein R ₁ and R ₂ is H or OCH ₃ Im).

The present invention provides a pharmaceutical composition for preventing or treating bone diseases, which comprises the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The scavenger of the present invention is characterized in that the scullocaproflavone derivative is scullocaproflavone I or scullocaproflavone II. In the scullocaflavone derivative of the present invention, each of R ₁ and R ₂ independently represents hydrogen (H) or methoxy It is selected from (OCH ₃₎ group.

In the formula (2), R < ₁ > and R < ₂ > are hydrogen (H).

In the formula ( ₃ ), R < ₁ > and R < ₂ > are methoxy (OCH3) groups.

In addition, the scalarcapoflavone derivative of the present invention preferably has a concentration of 0.1 to 10 μM. The scalarcaflavone derivative composition having a concentration of less than 0.1 μM has a problem of lowering the therapeutic efficiency of bone diseases, and when the concentration is more than 10 μM, there is a problem that toxicity can be produced in cells. In this respect, the concentration of the pharmaceutical composition containing the scalarcaflavone derivative as an active ingredient may preferably be 1 to 5 μM, more preferably 2 μM, but is not limited thereto.

In addition, the composition is characterized by decreasing the expression or activity of NFATc1, c-FOS, MAPKS, Src, AKT, CREB, PGC-1β and caveolin-1 and enhances the expression or activity of Nrf2, FOXO1 and catalase The present invention is characterized by inhibiting the production of active oxygen in the cell and inhibiting bone damage caused by inflammation.

In addition, bone diseases to which the composition of the present invention can be applied include osteoporosis caused by growth retardation, fracture, excessive bone resorption of osteoclasts, rheumatoid arthritis, periodontal disease, Paget disease, and metastatic bone cancers. However, the present invention is not limited thereto.

In a specific experimental example of the present invention, it was confirmed that the scalarcaflavone II (hereinafter referred to as SF II) of the present invention, which is the scaffold capsaicin derivative according to the present invention, effectively inhibited the differentiation of osteoclast precursor cells (BMMs) by RANKL (See FIG. 1). When SFII was treated, mRNA expression of CR and DC-STAMP genes was inhibited together with NFATc1 protein, which is the most important transcription factor in osteoclast differentiation, and CK, MMP9, and TRAP, which are the target genes thereof (see FIG. 2 ).

In addition, it was confirmed that the expression of c-Fos protein and mRNA, which are known as upstream regulators of NFATc1 in the presence of SF II, was significantly inhibited (see Fig. 3), confirming that phosphorylation of MAPKS, Src-AKT and FOXO1 was inhibited 4). Thus, the degradation of FOXO1 was also inhibited. As a result, the transcription activity of FOXO1 was increased and the expression of catalase, a target gene, was increased at both protein and mRNA levels (see FIG. 4).

In addition, it was confirmed that SFII significantly suppressed mRNA expression of CREB and PGC-1β and its target genes ND4, COX1, COX3 and Cyt b (see FIG. 5), and Nrf2 and representative target genes NQO1 and Srx MRNA expression was increased at the same time. At the same time, Nrf2 and Nrf2 target proteins including Nrf2 and antioxidant enzymes NQO1, Srx, PrxI, PrxII, PrxIII, PrxIV, PrxV, PrxVI, Trx1, Trx2, TrxR1 and TrxR2 were also increased. Also, it was confirmed that the generation of reactive oxygen species was reduced by SF II, and the expression of caveolin-1, which inhibits the activity of Nrf2 and is known as an osteoclast differentiation promoting factor, was also inhibited by SF II (see FIG. 6).

In addition, it was confirmed that SF II inhibited the formation of actin rings and osteoclast-induced bone resorption in dentine discs (see FIGS. 7 and 8), thereby inhibiting bone damage due to LPS inflammation (see FIG. 9).

Accordingly, the composition containing the scalarcaplavone derivative of the present invention as an active ingredient can be usefully used for prevention or treatment of bone diseases.

The present invention also encompasses not only the compounds represented by the above formula (1) but also pharmaceutically acceptable salts thereof, possible solvates, hydrates, racemates or stereoisomers thereof.

The present invention can be used in the form of a compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Derived from non-toxic organic acids, such as, for example, diesters, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinic acid, succinic acid, succinic acid, succinic acid, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluene sulfonate, chlorobenzene sulfoxide Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be obtained by a conventional method, for example, by dissolving the compound represented by the above formula (1) in an excess amount of an acid aqueous solution, and then mixing the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile ≪ / RTI > Further, it is also possible to prepare by heating the same amount of the compound represented by the above-mentioned formula (1) and an aqueous acid solution or alcohol, and then evaporating the mixture or drying the precipitated salt by suction filtration.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

When the composition is formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules, troches and the like, which may contain one or more excipients in the compound of formula (I) of the present invention, Starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included. have.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 81, cacao butter, taurine, glycerol, gelatin and the like.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the compound according to the present invention may vary depending on the age, sex, and body weight of the patient. In general, 0.1 mg to 100 mg, preferably 0.5 mg to 10 mg per kg of body weight is administered daily or every other day Or one to three times a day. However, the dosage may be varied depending on the route of administration, the severity of the disease, sex, weight, age, and the like, and therefore the dose is not limited to the scope of the present invention by any means.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The present invention also provides a health functional food for improving osteoporosis, which contains, as an active ingredient, a scalarcaplavone derivative represented by the general formula (1) or a pharmaceutically acceptable salt thereof.

In addition, the above-described scalarcaplavone derivative is characterized by being scalarcaplavone I or scalarcaplavone II.

The above-mentioned bone diseases include osteoporosis, rheumatoid arthritis, periodontal disease, Paget disease and metastatic bone cancer due to growth retardation, fracture, excessive bone resorption of osteoclasts (osteoporosis, rheumatoid arthritis, periodontal disease, metastatic bone cancers), but the present invention is not limited thereto.

As used herein, the term "health functional food" is produced by using raw materials or ingredients (functional raw materials) having functions useful for nutrients or human body that are likely to be deficient in daily eating, and is intended to maintain the normal function of the human body, But is not limited to, and is not meant to exclude health food in the usual sense.

The composition of the present invention can be added directly to food or used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). In general, the amount of the compound in the health functional food may be 0.01 to 90 parts by weight based on the total weight of the food. However, when consumed for a long period of time for the purpose of health and hygiene or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention is not particularly limited to other components other than those containing the above-mentioned composition as an essential ingredient at the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As a flavor other than the above, a natural flavoring agent {tau martin, stevia extract (for example, rebaudioside A, glycyrrhizin etc.)} and synthetic flavorings (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

These components may be used independently or in combination. The ratio of such additives is not so important, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail with reference to experimental examples.

However, the following experimental examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following experimental examples.

< Experimental Example  1> SF Ⅱ inhibition of osteoclast differentiation

Sculpaflavone II (hereinafter referred to as SF II) containing the present invention as an active ingredient was used in the experiment using ChemFaces CFN 92216 / 55084-08-7.

First, in order to confirm the inhibitory effect of SF II on osteoclast differentiation, the present inventors conducted experiments on BMM (bone marrow-derived macrophages) cells. BMM cells are precursor cells capable of differentiating into osteoclasts. In this experiment, cells from bone marrow of femur and tibia of 8-week-old C57BL / 6 male mice were used.

M-CSF (macrophage colony stimulating factor) at 25 ng / ml and RANKL at 50 ng / ml were injected into BMM (Bone Marrow-Derived Macrophages) cells in the presence of SF II at various concentrations (0, 1, 2 and 3 μM) The cells were stained with osteoclast, treated with PBS, fixed with 4% paraformaldehyde, and stained with TRAP using a leukocyte acid phosphatase cytochemistry kit (Sigma Aldrich). The number of osteoclasts with three or more TRAP stained cells was counted using a microscope Respectively.

As a result, osteoclast differentiation was effectively inhibited from 2 μM of SF II (FIGS. 1A and 1B).

In addition, cytotoxicity was analyzed with EASY Cytox (WST-1) assay kit to exclude the effect of SF II on osteoclast differentiation inhibition by cytotoxicity. BMM cells were treated with 10 (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium monosodium salt (WST- and incubated at 37 ° C for 2 - 4 hours, and the absorbance at 450 nm was measured.

As a result, it was confirmed that no cytotoxicity was observed by SF II up to a concentration of 3 μM (FIG. 1C). In addition, BMM cells were differentially treated with RANKL for 3 to 5 days in the presence of 2 μM SF II, and the osteoclast differentiation was significantly inhibited (D and E in FIG. 1) and no cytotoxicity was observed until 5 days (Fig. 1F). This suggests that the inhibitory effect of SF II on osteoclast differentiation is not due to apoptosis.

< Experimental Example  2> SF By II NFATc1  Inhibit activation

To confirm the inhibitory effect of SF II on osteoclast differentiation, BMM cells were treated with 50 ng / ml of RANKL for 2 days in the presence of SF II at various concentrations (0, 1, 2, 3 μM) to differentiate into osteoclasts, To confirm the expression of NFATc1 protein, the most important transcription factor in cell differentiation, immunoblot was performed using an antibody against NFATc1.

Proteins were separated using 10% polyacrylamide gel, and the separated proteins were transferred to nitrocellulose membranes. All non-specific binding sites in the membrane were blocked using 5% skim milk dissolved in TBST (0.05% Tween-20 in Tris-buffered saline, pH 7.4). The primary antibody (mouse monoclonal antibodies, Santa Cruz Biotechnology Inc.) was treated for 14 h at 4 ° C and the secondary antibody was treated for 1 h at room temperature and analyzed by West save Up (Ab Frontier).

As a result, the NFATc1 protein was significantly inhibited from the concentration of 2 μM SF II (FIG. 2A), and the inhibitory effect was maintained until 5 days (FIG. 2B).

In addition, changes in mRNA expression of the CK, MMP9 and TRAP genes, which are the target genes of NFATc1, and the DC-STAMP gene, which is involved in CR and osteoclast fusion, were confirmed by real time PCR.

For real-time PCR, total RNA was extracted using Trizol reagent (Invitrogen) and quantitated at 260 nm absorbance. The total RNA was loaded with 15 ml of oligo (dT) primers containing 0.5 mg After incubation for 5 minutes at &lt; RTI ID = 0.0 &gt; 0 C, &lt; / RTI &gt; The first strand of the cDNA was synthesized at 42 ° C for 1 hour and at 70 ° C for 10 minutes in a final volume of 25 μl containing 200 units of M-MLV reverse transcriptase (Promega), 24 units of ribonuclease inhibitor and 0.25 mM of each dNTP. In a final volume of 20 μl containing 0.8 μl of diluted cDNA template (1: 2.5), 10 μl of 2X SYBR Green PCR Master Mix (M Biotech) and 1 μl of each gene-specific primer (Genotect) 10 μM ABI 7300 real time PCR system (Applied Biosystems). Amplification was performed at 50 ° C for 2 minutes, at 95 ° C for 2 minutes, then at 95 ° C for 15 seconds, and at 60 ° C for 1 minute for 40 cycles. The sequences of the primers used in the experiments are shown in Table 1 below.

real-time PCR  The sequence of the primer and amplicon  size Gene
Primer (sense & antisense) SEQ ID NO: Amplicon length (bp) One NFATc1
5'-CTTCAGCTGGAGGACACC-3 ' One 79 5'-CCAATGAACAGCTGTAGCG-3 ' 2 2
CK
5'-ACCACTGCCTTCCAATACG-3 ' 3 99
5'-CGTGGCGTTATACATACAAC-3 ' 4 3
MMP9
5'-AGACGACATAGACGGCATC-3 ' 5 97
5'-TGCTGTCGGCTGTGGTTC-3 ' 6 4
TRAP
5'-CCAGCGACAAGAGGTTCC-3 ' 7 113
5'-AGAGACGTTGCCAAGGTGAT-3 ' 8 5
SOD2
5'-ATTAACGCGCAGATCATGCA-3 ' 9 161
5'-TGTCCCCCACCATTGAACTT-3 ' 10 6
COX2
5'-GATCATAAGCGAGGACCTG-3 ' 11 85
5'-GTCTGTCCAGAGTTTCACC-3 ' 12 7 c-Fos
5'-GAGAAACGGAGAATCCGAAG-3 ' 13 97
5'-GAGAAACGGAGAATCCGAAG-3 ' 14 8
PGC-1?
5'-CTCCAGGCAGGTTCAACCC-3 ' 15 83
5'-GGGCCAGAAGTTCCCTTAGG-3 ' 16 9
ND4
5'-CATCACTCCTATTCTGCCTAGCAA-3 '     17 74
5'-TCCTCGGGCCATGATTATAGTAC-3 '     18 10
COX1
5'-TTTTCAGGCTTCACCCTAGATGA-3 ' 19 81
5'-GAAGAATGTTATGTTTACTCCTACGAATATG-3 ' 20 11
COX3
5'-CGGAAGTATTTTTCTTTGCAGGAT-3 ' 21 82
5'-CAGCAGCCTCCTAGATCATGTG-3 ' 22 12
Cytb
5'-GCCACCTTGACCCGATTCT-3 ' 23 64
5'-TTGCTAGGGCCGCGATAAT-3 ' 24 13
Nrf2
5'-TCTCCTCGCTGGAAAAAGAA-3 ' 25 498
5'-AATGTGCTGGCTGTGCTTTA-3 ' 26 14 NQO1 5'-TTCTCTGGCCGATTCAGAG-3 ' 27 180
5'-GGCTGCTTGGAGCAAAATAG-3 ' 28 15 Srx 5'-AGCCTGGTGGACACGATC-3 ' 29 97
5'-AGGAATAGTAGTAGTCGCCA-3 ' 30 16
b-actin
5'-ACCCTAAGGCCAACCGTG-3 ' 31 81 5'-GCCTGGATGGCTACGTAC-3 ' 32 17
CR
5'-TGATGACTCTCAGGACAATG-3 ' 33 87 5'-ACTGGATCAATCTGTAGGAG-3 ' 34 18
DC-STAMP
5'-TTATGTGTTTCCACGAAGCCCTA-3 ' 35 141 5'-ACAGAAGAGAGCAGGGCAACG-3 ' 36 19
Catalase
5'-CTCGTTCAGGATGTGGTTTTC-3 ' 37 145 5'-CTTTCCCTTGGAGTATCTGGTG-3 ' 38 CK, cathepsin K; MMP9, matrix metalloprotease 9; SOD2, superoxide dismutase 2; COX2, cyclooxygenase 2

As a result, it was confirmed that the expression of CR and DC-STAMP gene mRNA was significantly suppressed in addition to NFATc1 and its target genes CK, MMP9 and TRAP when treated with 50 ng / ml RANKL for 2 days in the presence of 2 μM SFⅡ (FIG. 2C).

< Experimental Example  3> SF C- FOS  control

To determine whether SFII regulates NF-κB activity and c-Fos expression, known as upstream regulators of NFATc1, the degree of phosphorylation of IκBα was determined by immunoblotting with rabbit polyclonal antibodies (Cell Signaling Technology) against IκBα and p-IκBα The expression of SOD2 and c-FOS protein was confirmed by immunoblot using SOD2 / rabbit polyclonal antibody (Upstate; c-FOS / rabbit polyclonal antibody, Santa Cruz Biotechnology, Inc) mRNA expression was analyzed by real-time PCR as described in Experimental Example 2 (see Table 1, primer sequences).

As a result, SF II (2 μM) had no significant effect on the phosphorylation of IκBα in the presence of RANKL (50 ng / ml) (FIG. 3 A) and a significant effect on the protein and mRNA expression of SOD2, the NF- (B and D in Fig. 3). However, it was confirmed that SF II significantly inhibited the expression of c-Fos protein and mRNA (C and D in Fig. 3).

< Experimental Example  4> SF By II MAPKs  And AKT  Active inhibition

To investigate the effect of RANKL-activated MAPKs and of SFII on AKT, Rabbit polyclonal antibodies against these antibodies (phospho-JNK, phospho-ERK, phospho-p38, phospho-Src, phospho-AKT, phospho-Foxo1, antibodies, AKT, rabbit monoclonal antibodies against Foxo1, Rabbit polyclonal antibodies against JNK1, p38, ERK2 mouse monoclonal antibody, Santa Cruz Biotechnology, Inc.) compared the degree of phosphorylation of MAPKs and AKT-related proteins with immunoblot saw.

As a result, it was confirmed that phosphorylation of MAPKs and Src-AKT was inhibited by SFII (FIGS. 4A and 4B). In addition, since AKT is known to phosphorylate FOXO1 to induce degradation and inhibit migration to the nucleus, it was confirmed that addition of SFII inhibited the phosphorylation of FOXO1 (FIG. 4B) . SF II inhibited AKT activity and inhibited the phosphorylation of FOXO1, thereby confirming the inhibition of FOXO1 degradation (FIG. 4C). As a result, the transcription activity of FOXO1 was increased, and the expression of catalase, the target gene, was increased at both the protein and mRNA levels by immunoblot and real-time PCR (FIGS.

< Experimental Example  5> SF By II CREB? PGC - 1β  Active inhibition

PGC-1β is a well-known transcription factor that is activated by RANKL and is also involved in osteoclast differentiation. In order to confirm the effect of PGC-1β on SF II, mRNA expression of PGC-1 β and its target genes ND4, COX1, COX3 and Cyt b was confirmed by real-time PCR (see Table 1, primer sequence).

As a result, SF II (2 μM) significantly inhibited mRNA expression of PGC-1β, ND4, COX1, COX3 and Cyt b by RANKL (50 ng / ml) (FIG. In addition, SF II inhibited the expression of PGC-1β protein, and CREB (phosphobenzidine-2-phosphate dehydrogenase (CREB) monoclonal antibody, CREB mouse monoclonal antibody, which is the parent transcription factor of rabbit polyclonal antibody, Abcam , Inc.) (Fig. 5B and Fig. 5C). Indicating that SF II inhibits the CREB-PGC-1? Pathway.

< Experimental Example  6> SF By II Nrf2  Activation and active oxygen regulation

Since the redox-sensitive transcription factor Nrf2 is known to be involved in osteoclast differentiation by regulating the expression of GSH synthase and antioxidant enzyme in vivo, the effect of SF2 on osteoclast differentiation inhibition through the regulation of Nrf2 activity , Nrf2, NQO1 and Srx gene expression were confirmed by real-time PCR (see primer sequence in Table 1). Nrf2 and Nrf2 target proteins including Nrf2 and antioxidant enzymes NQO1, Srx, PrxI, PrxII, PrxIII, PrxIV, PrxV, Expression of Prx VI, Trx1, Trx2, TrxR1 and TrxR2 (rabbit polyclonal antibodies, Young In Frontier) protein was confirmed by immunoblot and real-time PCR.

As a result, mRNA expression of Nrf2 and its target genes NQO1 and Srx was increased by treatment with SF II (2 μM) in the presence of RANKL (50 ng / ml) (FIG. 6A). In addition, NQI1, Srx, PrxI, PrxII, PrxIII, PrxIV, PrxV, PrxVI, Trxl, Trx2, TrxRl and TrxR2, which are the target proteins including Nrf2 and antioxidant enzymes, were also found to be increased (Fig.

In addition, it is well known that RANKL-induced osteoclast differentiation is involved in the production of reactive oxygen species and also in the differentiation process. If the expression of Nrf2 target protein including antioxidant enzyme is increased by activation of Nrf2 by SFII, In order to confirm this possibility, RANKL was treated for 2 days in the presence of SF II and reactive oxygen species (ROS) were measured.

Measurement of ROS was carried out using oxidative-sensitive fluorescence detection using CM-H2DCFDA. CM-H2DCFDA reacted with ROS to generate fluorescence. Cells were treated with RANKL, washed with HBSS, treated with 5 μM CM-H2DCFDA for 10 min in the dark, and analyzed using a FACS Calibur flow cytometer (BD Biosciences) Respectively.

As a result, it was confirmed that the intracellular active oxygen was significantly decreased in the presence of SF II (left side of FIG. 6C).

SF II (2 μM) was pretreated for 30 minutes, and RANKL (C in FIG. 6) or H ₂ O ₂ (C in FIG. 6) , Right) was treated for 15 minutes, and then active oxygen was measured using CM-H2DCFDA as described above.

As a result, it was confirmed that the intracellular active oxygen was decreased by SF II (FIG. 6C, middle and right). Since SFII was pretreated only for a short time before the expression of antioxidant enzymes by Nrf2 activation, this result means that the active oxygen can be directly removed by SFII.

In addition, since caveolin-1 regulates the expression of Nrf2 and is known as a promoter of osteoclast differentiation, the effect of SFII on the expression of caveolin-1 has been investigated. As a result, it has been found that SFII inhibits caveolin-1 (rabbit monoclonal antibody, Trnasduction Laboratories) (FIG. 6D). This suggests that SFII inhibits the expression of caveolin-1 and reduces the binding to Nrf2 thereby promoting the activity of Nrf2 and consequently lowering intracellular reactive oxygen.

< Experimental Example  7> SF Ⅱ inhibition of osteoclast bone resorption

In order to investigate the timing of inhibition of osteoclast differentiation of SF II, BMM cells were treated with RANKL at various time points (E0, RANKL treatment; E1, RANKL treatment 1 day, E2, RANKL 2 day After treatment, the cells were exposed to SF II and analyzed for TRAP-stained multinuclear osteoclasts.

As a result, osteoclast formation was significantly inhibited when SF II (2 μM) was treated with RANKL (50 ng / ml), and when osteoclast formation was suppressed after RANKL 1 day treatment (E1) (E2) showed a significant difference in the number of osteoclasts over 10 nuclei, but it had little effect on the number of osteoclasts over 3 nuclei (Fig. 7A ).

Expression of NFATc1 and its target genes CK, MMP9, TRAP, CR and DC-STAMP were also measured by real-time PCR (see Table 1 primer sequences) and when SFII was treated with RANKL (E0) and SFII were significantly suppressed after treatment with RANKL 1 day (E1), but only the DC-STAMP gene was significantly affected by SFII (E2) exposed 2 days after treatment with RANKL (Fig. 7B).

In order to examine the effect of SFII on the formation of actin rings and bone resorption under similar experimental conditions, BMM cells were treated with RANKL for 3 days to differentiate into osteoclasts and treated with SF II and SF II Cells treated with RANKL were fixed as follows and stained with actin rings and nuclei or analyzed for bone resorption in dentine discs. Cells were fixed in 3.7% formaldehyde-supplemented PBS, treated with 0.1% Triton X-100, treated with Alexa Fluor 488-phalloidin (Invitrogen) for 20 minutes, and then treated with DAPI (4 ', 6-diamidino- , Roche) and observed with a fluorescence microscope. The degree of bone resorption was determined by removing cells from the dentine disc (Immunodiagnostic Systems Ltd) using a cotton tip, resampling the resorption pit with hematoxylin, and photographing it with a microscope at a magnification of 100 × using Image-Pro Plus 4.5 software (Media Cybernetics).

 As a result, when treated with RANKL, SFII completely inhibited the formation of actin rings and osteoclast-induced bone resorption in dentine discs, and when SFII was treated after RANKL treatment for 3 days and osteoclast differentiation It was confirmed that the size of the actin ring was reduced and the bone resorption was significantly inhibited (Figs. 8A, 8B and 8C). These results indicate that SF II can inhibit osteoclast fusion and bone resorption.

< Experimental Example  8> SF Ⅱ inhibition of bone damage by inflammation

In order to confirm the effect of inhibiting bone injury induced by SF II inflammation in animal models, the mouse skull was injected twice with LPS (Lipopolysaccharide, 12.5 mg / kg body weight) for 2 days at intervals. Five days after the first injection, the skull was fixed with 4% paraformaldehyde, followed by washing with PBS, followed by TRAP staining. The tumor was treated with vehicle (10% DMAC + 10% Tween 80 + 80% distilled water) or SF II (2 mg / kg) Respectively. In addition, the calcification was removed with 0.5 M ethylenediaminetetraacetic acid for 7 days, paraffin block was made, and then cut and stained with TRAP and hematoxilin.

As a result, TRAP-stained portions of the skulls treated with LPS alone were more abundant, but TRAP-stained portions were significantly decreased in the skulls treated with SF II (FIG. 9A). In addition, TRAP staining of the divided skulls after calcification removal showed that the number of bone cavities and osteoclasts increased by LPS treatment was considerably reduced by SF II (Fig. 9B and Fig. 9C).

<110> Ewha University - Industry Collaboration Foundation <120> Composition for prevention or treatment of bone disease          containing skullcap flavone derivatives or          acceptable salts thereof as an active ingredient <130> 2016P-03-043 <160> 38 <170> KoPatentin 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NFATc1 F <400> 1 cttcagctgg aggacacc 18 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NFATc1 R <400> 2 ccaatgaaca gctgtagcg 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CK F <400> 3 accactgcct tccaatacg 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CK R <400> 4 cgtggcgtta tacatacaac 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> MMP9 F <400> 5 agacgacata gacggcatc 19 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> MMP9 R <400> 6 tgctgtcggc tgtggttc 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TRAP F <400> 7 ccagcgacaa gaggttcc 18 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRAP R <400> 8 agagacgttg ccaaggtgat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOD2 F <400> 9 attaacgcgc agatcatgca 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOD2 R <400> 10 tgtcccccac cattgaactt 20 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> COX2 F <400> 11 gatcataagc gaggacctg 19 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> COX2 R <400> 12 gtctgtccag agtttcacc 19 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-Fos F <400> 13 gagaaacgga gaatccgaag 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> c-Fos R <400> 14 gagaaacgga gaatccgaag 20 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PGC-1 beta F <400> 15 ctccaggcag gttcaaccc 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PGC-1 beta R <400> 16 gggccagaag ttcccttagg 20 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ND4 F <400> 17 catcactcct attctgccta gcaa 24 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ND4 R <400> 18 tcctcgggcc atgattatag tac 23 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> COX1 F <400> 19 ttttcaggct tcaccctaga tga 23 <210> 20 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> COX1 R <400> 20 gaagaatgtt atgtttactc ctacgaatat g 31 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> COX3 F <400> 21 cggaagtatt tttctttgca ggat 24 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> COX3 R <400> 22 cagcagcctc ctagatcatg tg 22 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cytb F <400> 23 gccaccttga cccgattct 19 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cytb R <400> 24 ttgctagggc cgcgataat 19 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nrf2 F <400> 25 tctcctcgct ggaaaaagaa 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nrf2 R <400> 26 aatgtgctgg ctgtgcttta 20 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NQO1 F <400> 27 ttctctggcc gattcagag 19 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NQO1 R <400> 28 ggctgcttgg agcaaaatag 20 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Srx F <400> 29 agcctggtgg acacgatc 18 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Srx R <400> 30 aggaatagta gtagtcgcca 20 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Beta-actin F <400> 31 accctaaggc caaccgtg 18 <210> 32 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> beta-actin R <400> 32 gcctggatgg ctacgtac 18 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CR F <400> 33 tgatgactct caggacaatg 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CR R <400> 34 actggatcaa tctgtaggag 20 <210> 35 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> DC-STAMP F <400> 35 ttatgtgttt ccacgaagcc cta 23 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DC-STAMP R <400> 36 acagaagaga gcagggcaac g 21 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Catalase F <400> 37 ctcgttcagg atgtggtttt c 21 <210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Catalase R <400> 38 ctttcccttg gagtatctgg tg 22

Claims (11)

A pharmaceutical composition for preventing or treating bone diseases, which comprises, as an active ingredient, a scalarcaflavone derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof,
The bone diseases include osteoporosis, rheumatoid arthritis, periodontal disease, Paget disease and metastatic osteoclastic osteoarthritis caused by growth retardation, fracture, excessive osteoclast bone resorption, osteoporosis, rheumatoid arthritis, periodontal disease, Paget disease, bone cancers. &lt; RTI ID = 0.0 &gt;
[Chemical Formula 1]

.
(Wherein R &lt; ₁ &gt; and R &lt; ₂ &gt; are H or OCH ₃ )
The pharmaceutical composition for preventing or treating bone diseases according to claim 1, wherein the scalarcaplavone derivative is scalarcaplavone I or scalarcaplavone II.
The pharmaceutical composition for preventing or treating bone diseases according to claim 1, wherein the scaffold complex has a concentration of 0.1 to 10 μM.
The method of claim 1, wherein the composition is a pharmaceutical for the prevention and treatment of NFATc1, c-FOS, MAPK _S, Src, AKT, CREB, bone, characterized in that to reduce the PGC-1β and the expression or activity of caveolin-1 disease Gt;
The pharmaceutical composition for preventing or treating bone diseases according to claim 1, wherein the composition increases the expression or activity of Nrf2, FOXO1 and catalase.
The pharmaceutical composition for preventing or treating bone diseases according to claim 1, wherein the composition inhibits the production of intracellular active oxygen.
The pharmaceutical composition for preventing or treating bone diseases according to claim 1, wherein the composition inhibits inflammation-induced bone damage.
delete 1. A health functional food for improving bone diseases comprising, as an active ingredient, a scalarcaflavone derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof,
The bone diseases include osteoporosis, rheumatoid arthritis, periodontal disease, Paget disease and metastatic osteoclastic osteoarthritis caused by growth retardation, fracture, excessive osteoclast bone resorption, osteoporosis, rheumatoid arthritis, periodontal disease, Paget disease, bone cancers) for the treatment of osteoporosis.
[Chemical Formula 1]

.
(Wherein R &lt; ₁ &gt; and R &lt; ₂ &gt; are H or OCH ₃ )
The health functional food for improving bone diseases according to claim 9, wherein the scalarcafoflavone derivative is scalarcafoflavone I or scalarcopaflavone II.
delete

Images