KR101832351B1 - Composition for prevention or treatment of bone disease containing cinchonine or pharmaceutically acceptable salts thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis comprising synonin or a pharmaceutically acceptable salt thereof as an active ingredient. More particularly, the present invention relates to a pharmaceutical composition for preventing or treating osteoporosis, which comprises, when RANKL is administered to BMM cells in the presence of cinchonine, NFATc1 and its target genes associated with osteoclast differentiation, NF-κB and c-Fos, known as upstream regulators of NFATc1, PGC-1β, a transcription factor involved in mitochondrial biogenesis, and their target genes The expression of ERK is inhibited, the phosphorylation of ERK, Src-AKT and FOXO1 is inhibited, and osteoclast formation and bone resorption are inhibited. Thus, the synkinin of the present invention can be effectively used for the prevention or treatment of bone diseases .

Description

TECHNICAL FIELD The present invention relates to a composition for prevention or treatment of bone diseases containing cinchonine or a pharmaceutically acceptable salt thereof as an active ingredient,

The present invention relates to a pharmaceutical composition for preventing or treating bone diseases containing synonin 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 usually bone mass is 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.

On the other hand, cinchonine (CN) is an alkaloid derived from Cinchona bark, and its effect is known to be anti-obesity and anti-diabetic (Korean Patent Laid-Open Publication No. 0946641), but the synkonin of the present invention has an effect of regulating osteoclast differentiation, Have not been known until now.

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 synonin inhibits bone resorption by osteoclasts, The present invention has been accomplished by confirming that the present invention can be effectively used as a pharmaceutical composition.

It is an object of the present invention to provide a pharmaceutical composition containing synonin or a pharmaceutically acceptable salt thereof as an active ingredient 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 cinchonine or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food for improving bone diseases containing cinchonine or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition for preventing or treating osteoporosis comprising synonin or a pharmaceutically acceptable salt thereof of the present invention as an active ingredient inhibits important factors involved in osteoclast differentiation, thereby inhibiting osteoclast differentiation, 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-course effects of synonin (CN), a composition of the present invention, on the differentiation of RANKL-treated osteoclast precursor cells (BMMs)
A: TRAP staining of osteoclasts differentiated by RANKL in the presence of various concentrations of CN;
B: Analysis of TRAP-stained multinuclear osteoclast numbers of A above;
C: Cytotoxicity analysis by concentration of CN (M: M-CSF / M + R: M-CSF and RANKL);
D: TRAP staining of osteoclasts differentiated by RANKL for various times in the presence of CN;
E: Analysis of TRAP-stained polynuclear osteoclast number of D above; And
F: Cytotoxicity analysis of CN over time (Veh: Vehicle).
Figure 2 shows the effect of the composition of the present invention, synonin (CN) on the expression of NFATc1 and target genes of RANKL-treated osteoclast precursor cells (BMMs)
A: effect of CN concentration on expression of NFATc1;
B: time-dependent effect of CN on the expression of NFATc1; And
C: Effect of CN on mRNA expression of NFATc1 and target genes.
Figure 3 shows the effect of the composition of the present invention, synonin (CN) on NF-kB activity and c-FOS expression in RANKL-treated osteoclast precursor cells (BMMs)
A: Effect of CN on phosphorylation and degradation of IκBα;
B: Effect of CN on expression of NF-κB target gene;
C: Effect of CN on the expression of c-Fos; And
D: Effect of CN on mRNA expression of c-Fos and NF-kB target genes.
Fig. 4 shows the effect of MAPKs and Src? On osteoclast precursor cells (BMMs) treated with RANKL. (CN), a composition of the present invention for the phosphorylation of AKT and FOXO1: < RTI ID = 0.0 >
A: Effect of CN on phosphorylation of MAPKs;
B: the effect of CN on the phosphorylation of Src-AKT and FOXO1;
C: Effect of CN on expression of FOXO1 and catalase; And
D: catalase mRNA Expression.
Figure 5 shows the effect of synonin (CN), a composition of the present invention, on the activity of CREB-PGC-1 beta in RANKL-treated osteoclast precursor cells (BMMs)
A: Effect of CN on mRNA expression of PGC-1? And target genes;
B: effect of CN on the expression of PGC-1?; And
C: Effect of CN on phosphorylation of CREB.
FIG. 6 shows the effect of exposure time of synonin (CN), a composition of the present invention, on the osteoclast differentiation and expression of NFATc1 and PGC-1β and their target genes, and the effect of RANKL-treated osteoclast precursor cells (BMMs) (CN), which is a composition of the present invention, on the formation of an actin ring and the absorption of bone in a dentine disc:
A: Effect of time point of exposure of CN on osteoclast differentiation;
B: Effect of exposure time point of CN on mRNA expression of NFATc1 and target genes;
C: Effect of exposure time of CN on mRNA expression of PGC-1? And target genes;
D: a diagram showing the experimental design of the exposure point of CN;
E: effect of CN exposure time on formation of actin ring; And
F: Effect of time point of exposure of CN on bone resorption.
Figure 7 is a graph showing the inhibitory effect of synonin (CN), a composition of the present invention, on bone damage caused by LPS-induced inflammation;
A: Photographs of the skull sections stained with TRAP and Hematoxilin; And
B: Analysis of the bone cavity and number of osteoclasts in A above.

Hereinafter, the present invention will be described in detail.

Cinchonine (CN) contained in the present invention as an active ingredient is an alkaloid derived from Cinchona bark and has a structure represented by the following formula (1):

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

The inventive shinkonin is characterized by a concentration of 0.1 to 50 μM. Synconein compositions having a concentration of less than 0.1 μM have a problem of lowering the therapeutic efficiency of bone diseases, and when the concentration is more than 50 μM, there is a problem that the cells are toxic. In this respect, the concentration of the pharmaceutical composition containing cinchonine as an active ingredient may be preferably 1 to 30 μM, more preferably 20 μM.

The composition is characterized by decreasing the expression or activity of NFATc1, NF-κB, ERK, Src, AKT, FOXO1, c-FOS and PGC-1β and is characterized by inhibiting bone damage due to 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 synonin (CN) of the present invention described in the above formula (1) effectively inhibited osteoclast differentiation by RANKL (see FIG. 1). When shinkonin was treated, mRNA expression of the NFATc1 protein, the most important transcription factor in osteoclast differentiation, and its target genes, CK, MMP9 and TRAP genes, was inhibited (see FIG. 2).

In addition, phosphorylation of IκBα was inhibited in the presence of synonin, and the expression of NF-κB and its target genes, SOD2 and COX2, also known as upstream regulators of NFATc1, were also inhibited. In addition, it was confirmed that c-Fos expression was significantly inhibited by cinchonine (see FIG. 3), and phosphorylation of ERK, Src-AKT and FOXO1 was inhibited (see FIG. 4).

In addition, it was confirmed that synonin inhibits mRNA expression of PGC-1β and its target genes ND4, Cytb, COX1 and COX3 (see FIG. 5), and it was confirmed that synonin inhibited the formation of actin ring and osteoclast- Inhibited bone resorption (see FIG. 6), and inhibited bone damage caused by LPS inflammation (see FIG. 7).

Therefore, the composition containing cinchonine (CN) 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, , 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 bone diseases, which comprises the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The above-mentioned bone diseases include osteoporosis, rheumatoid arthritis, periodontal disease, Paget disease and metastatic osteoclastic osteoclast due to growth retardation, fracture, excessive osteoclast bone resorption, osteoporosis, rheumatoid arthritis, periodontal disease, Paget disease, bone cancers, and the like. However, 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> Shinkonin  Osteoclast differentiation inhibition

Shinononin (hereinafter CN) containing the present invention as an active ingredient was used in the experiment using Sigma-Aldrich's 27370 / 118-10-5 product.

First, in order to confirm the inhibitory effect of synonin 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) and RANKL (50 ng / ml) were added to BMM (Bone Marrow-Derived Macrophages) cells in the presence of synonin at various concentrations (0, 10, 20 and 30 μM) PeproTech Inc.) was treated with PBS for 3 days and then fixed with 4% paraformaldehyde, followed by TRAP staining using leukocyte acid phosphatase cytochemistry kit (Sigma Aldrich), followed by TRAP staining with 3 or more polynuclear The number of osteoclasts was analyzed under a microscope.

As a result, osteoclast differentiation was effectively inhibited from 20 mu M of synonin (Fig. 1, A and B).

In addition, cytotoxicity was analyzed with the EASY Cytox (WST-1) assay kit to exclude the effect of cycononin on osteoclast differentiation by cytotoxicity. BMM cells were treated with 10 [mu] l of 4-nitrophenyl-5- (2,4-disulfophenyl) -2H-tetrazolium mono-sodium salt (WST-1) reagent (Roche Applied Science) and incubated at 37 ° C for 2 - 4 hours, and the absorbance at 450 nm was measured.

As a result, it was confirmed that cytotoxicity caused by synonin did not occur up to a concentration of 30 μM (FIG. 1C). In addition, when BMM cells were treated with RANKL for 3 to 5 days in the presence of 20 μM synonin, osteoclast differentiation was significantly inhibited (D and E in FIG. 1), and cytotoxicity was observed up to 5 days (Fig. 1F). This suggests that the effect of synonin on osteoclast differentiation is not due to apoptosis.

< Experimental Example  2> Shinkonin  by NFATc1  Inhibit activation

In order to confirm the inhibitory effect of synonin on osteoclast differentiation, BMM cells were treated with 50 ng / ml of RANKL for 2 days in the presence of synonin at various concentrations (0, 10, 20, 30 μM) to differentiate into osteoclasts In order to confirm the expression of NFATc1 protein, the most important transcription factor in osteoclast differentiation, immunoblot using an antibody against NFATc1 was performed.

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 20 μM synonin (FIG. 2A), and the inhibitory effect was maintained until 5 days (FIG. 2B).

In addition, mRNA expression changes of CK, MMP9 and TRAP genes, which are the target genes of NFATc1, were confirmed using real time PCR.

For real time PCR, total RNA was extracted using Trizol reagent (Invitrogen), quantitated at 260 nm absorbance, total RNA was added to a total of 15 μl, including 0.5 mg oligo (dT) primers, 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
β-actin
5'-ACCCTAAGGCCAACCGTG-3 ' 25 81 5'-GCCTGGATGGCTACGTAC-3 ' 26 14 Catalase
5'-CTCGTTCAGGATGTGGTTTTC-3 ' 27 145 5'-CTTTCCCTTGGAGTATCTGGTG-3 ' 28 CK, cathepsin K; MMP9, matrix metalloprotease 9; SOD2, superoxide dismutase 2; COX2, cyclooxygenase 2

As a result, it was confirmed that the expression of mRNA of NFATc1 and its target genes CK, MMP9 and TRAP genes was remarkably suppressed when treated with 50 ng / ml RANKL for 2 days in the presence of 20 μM synonin (FIG. 2C ).

Experimental Example 3 Synuclein Regulation of NF-κB and c-FOS

To determine whether synonin regulates NF-κB activity and c-Fos expression, known as upstream regulators of NFATc1, the degree of phosphorylation of IκBα by immunoblot using antibodies against IκBα and p-IκBα (Rabbit polyclonal antibodies, Cell Signaling Technology) SOD2 and c-Fos mRNA expression was also confirmed by immunoblot using SOD2 / rabbit polyclonal antibody (Upstate, c-FOS / Rabbit polyclonal antibody, Santa Cruz Biotechnology Inc.) And analyzed by real-time PCR according to the method described in Experimental Example 2 (see Table 1, primer sequence).

As a result, shinkonin (20 μM) significantly inhibited the phosphorylation of IκBα in the presence of RANKL (50 ng / ml) (FIG. 3 A), and the protein and mRNA expression of the NF-κB target genes SOD2 and COX2 (B and D in Fig. 3). In addition, it was confirmed that synonin significantly inhibited the expression of c-Fos protein and mRNA (C and D in Fig. 3).

<Experimental Example 4> Inhibition of ERK and Src-AKT activity by synonin

To investigate the effect of synkinin on MAPKs and AKT activated by RANKL, we examined the effects of these antibodies (phospho-JNK, phospho-ERK, phospho-p38, phospho-Src, phospho-AKT, phospho-Foxo1, The immunoprecipitation of MAPKs and AKT-related proteins with immunoblot using rabbit monoclonal antibodies against polyclonal antibodies, rabbit monoclonal antibodies against Foxo1, Rabbit polyclonal antibodies against JNK1 and p38, and ERK2 mouse monoclonal antibody, Santa Cruz Biotechnology, I compared them.

As a result, it was confirmed that phosphorylation of ERK and Src-AKT among MAPKs was suppressed by synonin (Figs. 4A and 4B). In addition, since AKT is known to phosphorylate FOXO1 to induce degradation and inhibit its migration to the nucleus, it was confirmed that addition of synonin affects the phosphorylation of FOXO1, as a result, it was confirmed that FOXO1 inhibited phosphorylation (FIG. 4B ). Shinkonin inhibited the phosphorylation of FOXO1 by inhibiting AKT activity, 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> Inhibition of PGC-1β activity by cinchonin

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

As a result, shinkonin (20 μM) significantly inhibited PGC-1β, ND4, Cyt b, COX1 and COX3 mRNA expression by RANKL (50 ng / ml) (FIG. In addition, synonin inhibited the expression of PGC-1β (rabbit polyclonal antibody, Abcam) protein, but CREB (phosphobenz-2-yl) phosphatidylinositol (CREB) monoclonal antibody Biotechnology, Inc) (Fig. 5, B and C).

< Experimental Example  6> Shinkonin  Of bone resorption by osteoclasts

In order to investigate the timing of inhibition of the differentiation of osteoclast by osteoclast differentiation, BMM cells were treated with RANKL at various time points (E0, RANKL treatment; E1, RANKL 1 day treatment; E2, RANKL 2 After treatment, the cells were exposed to cinchonin and the number of multicellular osteoclasts stained with TRAP was analyzed.

As a result, osteoclast formation was almost inhibited when synonin (20 μM) was treated with RANKL (50 ng / ml) (E0), and when synonin was treated after RANKL 1 day treatment However, treatment with synonin after 2 days (E2) showed little effect on osteoclast formation (Fig. 6A).

The expression of NFATc1 and its target genes CK, MMP9, TRAP and PGC-1β and their target genes ND4, COX1, COX3 and Cyt b were also measured by real-time PCR primer sequence). When synonin was treated with RANKL (E0), the expression was largely inhibited, but almost no inhibition was observed by synonin exposed after (E2) treatment with RANKL 2 days B and C).

To investigate the effects of synonin 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, Cells treated with cinchonin were treated with RANKL as follows, 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, shinkonin inhibited the formation of actin ring and bone resorption, but did not inhibit actin ring formation and bone resorption in cells differentiated with RANKL for 3 days (Fig. 6 D and F).

These results show that synonin can inhibit osteoclast formation and bone resorption.

<Experimental Example 7> Suppression of bone damage by inflammation of synonin

In order to confirm the effect of inhibiting bone damage induced by sinoninin in animal models, the mouse skull was injected with LPS (Lipopolysaccharide, 12.5 mg / kg body weight) twice daily at an interval of one day. Vehicle (20% polyethylene glycol + 20% ethanol + 60% distilled water) or shinkonin (10 mg / kg) was administered together. After 5 days of the first injection, the skull was fixed with 4% paraformaldehyde and washed with PBS. After removing the calcification with 0.5 M ethylenediaminetetraacetic acid for 7 days, paraffin blocks were cut and stained with TRAP and hematoxilin.

As a result, it was confirmed that the number of bone cavities and osteoclasts increased by LPS induction was considerably reduced by synonin (Figs. 7A and 7B).

<110> Ewha University - Industry Collaboration Foundation <120> Composition for prevention or treatment of bone disease          containing cinchonine or pharmaceutically acceptable salts          thereof as an active ingredient <130> 2016P-04-041 <160> 28 <170> Kopatentin 2.0 <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> 18 <212> DNA <213> Artificial Sequence <220> <223> Beta-actin F <400> 25 accctaaggc caaccgtg 18 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> beta-actin R <400> 26 gcctggatgg ctacgtac 18 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Catalase F <400> 27 ctcgttcagg atgtggtttt c 21 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Catalase R <400> 28 ctttcccttg gagtatctgg tg 22

Claims (7)

The present invention relates to a pharmaceutical composition for preventing osteoporosis, osteoporosis, rheumatoid arthritis, periodontal disease, osteoporosis, osteoporosis, osteoporosis, osteoporosis, osteoporosis, and Paget disease. The pharmaceutical composition for preventing or treating bone diseases according to any one of claims 1 to 3,
[Chemical Formula 1]

.
The pharmaceutical composition for preventing or treating bone diseases according to claim 1, wherein the synonin is in a concentration of 0.1 to 50 μM.
The pharmaceutical composition according to claim 1, wherein the composition reduces the expression or activity of NFATc1, NF-κB, ERK, Src, AKT, FOXO1, c-FOS and PGC-1β Composition.
The pharmaceutical composition for preventing or treating bone diseases according to claim 1, wherein the composition inhibits inflammation-induced bone damage.
delete The present invention relates to a pharmaceutical composition for preventing osteoporosis, osteoporosis, rheumatoid arthritis, periodontal disease, osteoporosis, osteoporosis, osteoporosis, osteoporosis, osteoporosis, disease and paget disease. The present invention relates to a health functional food for improving bone diseases,
[Chemical Formula 1]

.
delete

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