WO2018016901A1 - Pharmaceutical composition for preventing or treating bone diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating bone diseases and, specifically, the pharmaceutical composition containing Euphorbia factor or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating bone diseases of the present invention inhibits important factors involved in osteoclast differentiation, thereby inhibiting osteoclast differentiation to suppress bone resorption. Furthermore, the pharmaceutical composition of the present invention containing a skullcapflavone derivative or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating bone diseases inhibits important factors involved in osteoclast differentiation, thereby suppressing bone resorption. Furthermore, a pharmaceutical composition of the present invention containing dehydrocostus lactone or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating bone diseases inhibits important factors involved in osteoclast differentiation, thereby inhibiting osteoclast differentiation to suppress bone resorption. Furthermore, a pharmaceutical composition of the present invention containing cinchonine or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating bone diseases inhibits important factors involved in osteoclast differentiation, thereby inhibiting osteoclast differentiation to suppress bone resorption. Therefore, the compositions can be favorably utilized in preclinical and clinical studies and can treat bone-related diseases caused by an imbalance of osteoclasts, and thus can be widely utilized for the development of medicines for the prevention or treatment of bone-related diseases.

Description

Pharmaceutical composition for the prevention or treatment of bone disease

The present invention relates to a pharmaceutical composition for the prevention or treatment of bone diseases.

Osteoporosis, a representative metabolic bone disease, is a disease in which bone mineral density is reduced and bone density becomes thinner, resulting in widening of the bone marrow cavity. It is easy to fracture even in shock.

Osteoporosis is characterized by a decrease in bone mass and abnormal microstructures. As aging progresses, the balance between the absorption of old bone and the formation of new bone is compromised, resulting in poor bone remodeling, which leads to bone failure. As a result, the risk of breakage or fracture increases. This bone mass is influenced by several factors, including genetic factors, nutrition, hormonal changes, differences in exercise and lifestyle, and age, lack of exercise, low weight, smoking, low calcium diet, menopause and ovarian ablation. It is a major cause of bone loss.

On the other hand, although there are individual differences, bone mass is usually the highest at 14-18 years of age, and decreases by about 1% per year in old age. In particular, women after 30 years of bone reduction continues to progress, and by the hormonal changes, bone reduction rapidly progresses. In other words, estrogen concentration rapidly decreases at the end of menopause, in which a large amount of B-lymphocytes are produced, as in the case of IL-7 (interleukin-7), and the B cell precursor (pre-B) is added to the bone marrow. cells) accumulate, thereby increasing the amount of IL-6, increasing the activity of osteoclasts, and eventually reducing bone mass.

As such, osteoporosis is an unavoidable symptom for elderly people, especially postmenopausal women, although the degree of osteoporosis is increasing, and as the population ages in developed countries, interest in osteoporosis and its therapeutics is gradually increasing. In addition, around $ 130 billion is being created around the world in the treatment of bone disease, and because it is expected to grow further, research institutions and pharmaceutical companies around the world are investing heavily in developing bone disease treatments.

In Korea, the prevalence of osteoporosis is rapidly increasing as the average life expectancy reaches 80. Recently, a study of local residents showed that 4.5% of men and 19.8% of women had osteoporosis when standardized to the national population. It is reported to have. This suggests that osteoporosis is more common than diabetes or cardiovascular disease, and osteoporosis is a very important health problem when estimating the pain or cost of treating patients who suffer from fractures.

To date, several substances have been developed for the treatment of osteoporosis. Among them, estrogen, which is most commonly used as a therapeutic agent for osteoporosis, has not yet been tested for its actual efficacy and has to be taken continuously throughout life, and has long-term side effects such as increased breast cancer or uterine cancer. Alendronate also has a problem that the effect is not clear, slow absorption in the digestive tract and inflammation of the stomach and esophagus mucosa. Calcium preparations are known to have fewer side effects and superior effects, but they are more preventive agents than therapeutic agents. Other vitamin D preparations, such as calcitonin, are known but have not been fully studied for their efficacy and side effects. Accordingly, there is a need for a new metabolic bone disease treatment agent having fewer side effects and excellent effects.

Osteoclasts are large multinucleated cells that destroy or absorb bone tissue that has become unnecessary during the growth of bones of vertebrates and are differentiated from osteoclast precursors. Osteoclast progenitor cells differentiate into osteoclasts in the presence of M-CSF and RANKL, and form multinucleated osteoclasts through fusion. Osteoclasts bind to bone through αvβ3 integrin and create an acidic environment while secreting various collagenases and proteases, resulting in bone resorption. Can be an effective way of treating bone disease.

Meanwhile, Euphorbia Factor L1 (hereinafter referred to as EFL1) is a diterpenoid obtained from seeds of Euphorbia lathyris linne (responder / poor), antiseptic, anticancer and apoptosis sensitization. ) Is known (Zang JY. Et al., Molecules, 2013, 18, 12793-12808). In addition, the Republic of Korea Patent Publication No. 2001-0028373 discloses the use of EFL1 as a drug or cosmetic material as a keratin exfoliation promoter, the osteoclast differentiation control effect of EFL1 of the present invention and the purpose of treating bone disease is not known to date.

In addition, the scullcapflavone derivative is a flavonoid derived from Scutellaria baicalensis (gold), which is known to have anti-inflammatory and anticancer effects by folk remedies. Thus, the effect of inhibiting allergic asthma is known (Yun-Choi. Et al., Archives of pharmacal research, v. 16 no. 4, 1993, 283-288). In addition, the Republic of Korea Patent Publication 2013-0120849 discloses the use of the skull cap flavone derivatives as a pharmaceutical material as a composition for the prevention or treatment of asthma, but the osteoclast differentiation control effect of the skull cap flavone derivatives of the present invention and the purpose of treating bone diseases None known until now.

In addition, dehydrocostus lactone (DHCL) is known to inhibit NF-κB activiation by TNFα and to increase the activity of caspase3 and caspase8 to promote apoptosis. Although contained in the back and the like, the protective effect of hepatocytes against oxidative stress is known, but the osteoclast differentiation regulating effect of the present invention and the purpose of treating bone disease is not known until now.

In addition, cinchonine (cinchonine, CN) is an alkaloid derived from Cinchona bark, but the effects of anti-obesity and anti-diabetic are known (Korean Patent Publication No. 0946641), the effect of controlling the differentiation of osteoclast differentiation and bone diseases of the present invention Is unknown to date.

Accordingly, the present inventors, while searching for a substance that inhibits the activity of osteoclasts in search of a new substance for treating bone disease, found that EFL1 inhibited the bone resorption by osteoclasts. It has been confirmed that the composition can be usefully used. In addition, it has been found that the skull capflavone derivatives inhibit bone uptake by osteoclasts, thereby confirming that the skull capflavone derivatives may be usefully used as pharmaceutical compositions for treating bone diseases. In addition, it was found that dihydrocostus lactone inhibits bone resorption by osteoclasts, and it was confirmed that dihydrocostus lactone may be usefully used as a pharmaceutical composition for treating bone diseases. In addition, the present invention was completed by confirming that synconin inhibits bone resorption by osteoclasts, and confirms that synconin may be usefully used as a pharmaceutical composition for treating bone diseases.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone diseases.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases containing Eupovia factor L1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention provides a dietary supplement for the improvement of bone diseases containing Euphorbia factor or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention or treatment of bone diseases containing a scalcap flavone derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention provides a health functional food for the improvement of bone disease containing a skull cap flavone derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention or treatment of bone diseases containing dihydrocostus lactone or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention provides a dietary supplement for the improvement of bone diseases containing dihydrocostus lactone or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases, which contains as a active ingredient cinconin or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a dietary supplement for the improvement of bone diseases, which contains as a active ingredient Cynconin or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition for preventing or treating bone diseases containing Euphorbia Factor of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient inhibits important factors related to osteoclast differentiation, thereby differentiating osteoclasts. There is an effect of inhibiting bone absorption by inhibiting.

In addition, the pharmaceutical composition for the prevention or treatment of bone diseases containing the skull capflavone derivative or a pharmaceutically acceptable salt thereof of the present invention to inhibit bone absorption by inhibiting important factors associated with osteoclast differentiation It works.

In addition, the pharmaceutical composition for the prevention or treatment of bone diseases containing the dihydrocostus lactone of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient, by inhibiting important factors associated with osteoclast differentiation, differentiation of osteoclasts There is an effect of inhibiting bone absorption by inhibiting.

In addition, the pharmaceutical composition for the prevention or treatment of bone diseases, including the present mykonin or a pharmaceutically acceptable salt thereof as an active ingredient inhibits the differentiation of osteoclasts by inhibiting important factors related to osteoclast differentiation Because of the effects of inhibiting bone absorption, the compositions can be usefully used for preclinical and clinical studies, and can treat bone-related diseases caused by the imbalance of osteoclasts, and thus are widely used for the prevention or treatment of bone-related diseases. Can be utilized.

1 is a diagram showing the concentration and time-dependent effects of the composition of the present invention EFL1 on the differentiation of RANKL treated osteoclast progenitor cells (BMMs) according to the concentration of EFL1:

A: TRAP staining of osteoclasts differentiated by RANKL in the presence of varying concentrations of EFL1;

B: analysis of the number of TRAP stained multinucleated osteoclasts of A;

C: concentration-specific cytotoxicity assay of EFL1 (M: M-CSF / M + R: M-CSF and RANKL);

D: TRAP staining of osteoclasts differentiated by RANKL for various times in the presence of EFL1;

E: Analysis of the number of TRAP stained multinucleated osteoclasts of D above; And

F: hourly cytotoxicity assay of EFL1 (Veh: Vehicle).

2 is a diagram showing the effect of EFL1, a composition of the present invention, on the expression of NFATc1 and target genes in RANKL treated osteoclast progenitor cells (BMMs):

A: concentration-dependent effects of EFL1 on the expression of NFATc1;

B: hourly effect of EFL1 on the expression of NFATc1; And

C: Effect of EFL1 on mRNA expression of NFATc1 and target genes.

3 is a diagram showing the effect of EFL1, the composition of the present invention on NF-κB activity and c-FOS expression in RANKL treated osteoclast progenitor cells (BMMs):

A: effect of EFL1 on phosphorylation and degradation of IκBα;

B: effect of EFL1 on expression of NF-κB target gene;

C: effect of EFL1 on the expression of c-Fos; And

D: Effect of EFL1 on mRNA expression of c-Fos and NF-κB target genes.

Figure 4 is a diagram showing the effect of EFL1 composition of the present invention on the phosphorylation of MAPKs and AKT in RANKL-treated osteoclast progenitor cells (BMMs).

5 is a diagram showing the effect of EFL1, the composition of the present invention on the activity of CREB-PGC-1β in RANKL treated osteoclast progenitor cells (BMMs):

A: effect of EFL1 on mRNA expression of PGC-1β and target genes;

B: effect of EFL1 on expression of PGC-1β; And

C: Effect of EFL1 on phosphorylation of CREB.

FIG. 6 is a diagram showing the effect of EFL1, a composition of the present invention, on the activity and production of free radicals of Nrf2 and its target genes in RANKL treated osteoclast progenitor cells (BMMs):

A: effect of EFL1 on mRNA expression of Nrf2 and target genes, and effect of EFL1 on free radical accumulation in cells; And

B: Effect of EFL1 on the expression of target proteins including Nrf2 and antioxidant enzymes.

FIG. 7 is a diagram showing the effects of exposure to EFL1, the composition of the present invention, on osteoclast differentiation and expression of NFATc1 and PGC-1β, and their target genes:

A: effect of EFL1 on exposure to osteoclast differentiation;

B: Effect of time of exposure of EFL1 on mRNA expression of NFATc1 and target genes; And

C: Effect of EFL1 on exposure to mRNA expression of PGC-1β and target genes.

FIG. 8 is a diagram showing the effects of the exposure time point of EFL1, the composition of the present invention, on actining formation of RANKL-treated osteoclast progenitor cells (BMMs) and bone resorption on dentine discs:

A: Chart showing the experimental design of the exposure time point of EFL1;

B: effect of time of exposure of EFL1 on the formation of actining; And

C: Effects of EFL1 on bone absorption at different time points.

9 is a diagram showing the effect of EFL1, the composition of the present invention on the death of osteoclasts:

A: Morphological changes of differentiated osteoclasts with treatment time of EFL1;

B: cytotoxicity assay of EFL1 in differentiated osteoclasts;

C: effect of EFL1 on the death of differentiated osteoclasts; And

D: Graph of C.

10 is a diagram showing the inhibitory effect of the composition of the present invention EFL1 against bone damage caused by LPS-induced inflammation:

A: Photograph of skull sections stained with TRAP and Hematoxilin; And

B: Analysis of the number of bone cavity and osteoclasts of A.

11 is a diagram showing the concentration and time-dependent effect of SFII, the composition of the present invention on the differentiation of RANKL-treated osteoclast progenitor cells (BMMs):

A: TRAP staining of osteoclasts differentiated by RANKL in the presence of various concentrations of SFII;

B: analysis of the number of TRAP stained multinucleated osteoclasts of A;

C: cytotoxicity analysis by concentration of SFII (M: M-CSF / M + R: M-CSF and RANKL);

D: TRAP staining of osteoclasts differentiated by RANKL for various times in the presence of SFII;

E: Analysis of the number of osteoclasts of multinucleated stained TRAP staining of D; And

F: hourly cytotoxicity assay of SFII (Veh: Vehicle).

12 is a diagram showing the effect of SFII, the composition of the present invention on the expression of NFATc1 and target genes in RANKL treated osteoclast progenitor cells (BMMs):

A: concentration-dependent effects of SFII on the expression of NFATc1;

B: hourly effect of SFII on the expression of NFATc1; And

C: Effect of SFII on mRNA expression of NFATc1 and target genes.

13 is a diagram showing the effect of SFII, the composition of the present invention on NF-κB activity and c-FOS expression in RANKL treated osteoclast progenitor cells (BMMs):

A: effect of SFII on phosphorylation and degradation of IκBα;

B: effect of SFII on expression of NF-κB target gene;

C: effect of SFII on the expression of c-Fos; And

D: Effect of SFII on mRNA expression of c-Fos and NF-κB target genes.

Figure 14 shows the effect of SFII, the composition of the present invention on phosphorylation of MAPKs and Src-AKT and FOXO1 in RANKL treated osteoclast progenitor cells (BMMs):

A: effect of SFII on phosphorylation of MAPKs;

B: effect of SFII on phosphorylation of Src-AKT and FOXO1;

C: effect of SFII on FOXO1 and catalase expression; And

D: Effect of SFII on mRNA expression of catalase.

15 is a diagram showing the effect of SFII, the composition of the present invention on the activity of CREB-PGC-1β in RANKL treated osteoclast progenitor cells (BMMs):

A: effect of SFII on mRNA expression of PGC-1β and target genes;

B: effect of SFII on expression of PGC-1β; And

C: Effect of SFII on phosphorylation of CREB.

FIG. 16 is a diagram showing the effect of SFII, a composition of the present invention, on the activity of Nrf2 and its target genes, caveolin-1 and production of free radicals in RANKL treated osteoclast progenitor cells (BMMs):

A: effect of SFII on mRNA expression of Nrf2 and target genes;

B: effect of SFII on the expression of target proteins including Nrf2 and antioxidant enzymes;

C: effect of SFII on free radical accumulation in cells; And

D: Effect of SFII on the expression of caveolin-1.

FIG. 17 is a diagram showing the effects of exposure time of SFII, a composition of the present invention, on osteoclast differentiation and expression of NFATc1 and its target genes:

A: Effect of SFII on osteoclast differentiation by time of exposure; And

B: Effect of SFII upon exposure to mRNA expression of NFATc1 and target genes.

FIG. 18 is a diagram showing the effects of SFII, the composition of the present invention, on the actining formation of RANKL-treated osteoclast progenitor cells (BMMs) and bone resorption on dentine discs:

A: chart showing experimental design of exposure time of SFII;

B: effect of time of exposure of SFII on the formation of actining; And

C: Effects of SFII on Bone Absorption by Time of Exposure.

19 is a diagram showing the effect of SFII, a composition of the present invention, on bone damage caused by LPS-induced inflammation:

A: photo of TRAP stained skull;

B: photograph of the skull section stained with TRAP and Hematoxilin; And

C: Analysis of the number of bone cavity and osteoclast of B.

20 shows the concentration and time-dependent effects of the composition of the present invention, dihydrocostus lactone (hereinafter referred to as DHCL) on the differentiation of RANKL-treated osteoclast progenitor cells (BMMs) according to the concentration of dihydrocostus lactone (DHCL). Is a diagram representing:

A: TRAP staining of osteoclasts differentiated by RANKL 4 day treatment in the presence of various concentrations of DHCL;

B: analysis of the number of TRAP stained multinucleated osteoclasts of A;

C: cytotoxicity assay by concentration of DHCL (M: M-CSF / M + R: M-CSF and RANKL);

D: TRAP staining of osteoclasts differentiated by RANKL 5 day treatment in the presence of various concentrations of DHCL;

E: Analysis of the number of osteoclasts of multinucleated stained TRAP staining of D; And

F: Cytotoxicity analysis by concentration of DHCL (M: M-CSF / M + R: M-CSF and RANKL).

FIG. 21 is a diagram showing the effect of dehydrocostus lactone (DHCL), a composition of the present invention, on the expression of NFATc1 and target genes in RANKL treated osteoclast progenitor cells (BMMs):

A: concentration-dependent effect of DHCL on expression of NFATc1;

B: hourly effect of DHCL on expression of NFATc1; And

C: Effect of DHCL on mRNA expression of NFATc1 and target genes.

FIG. 22 is a diagram showing the effect of dihydrocostus lactone (DHCL), a composition of the present invention, on NF-κB activity and c-FOS expression in RANKL treated osteoclast progenitor cells (BMMs):

A: effect of DHCL on phosphorylation and degradation of IκBα;

B: effect of DHCL on expression of NF-κB target gene;

C: effect of DHCL on expression of c-Fos; And

D: Effect of DHCL on mRNA expression of c-Fos and NF-κB target genes.

FIG. 23 is a diagram showing the effect of dihydrocostus lactone (DHCL), a composition of the present invention, on phosphorylation of MAPKs and Src-AKT and FOXO1 in RANKL treated osteoclast progenitor cells (BMMs):

A: effect of DHCL on phosphorylation of MAPKs;

B: effect of DHCL on phosphorylation of Src-AKT and FOXO1;

C: effect of DHCL on FOXO1 and catalase expression; And

D: Effect of DHCL on mRNA expression of catalase.

FIG. 24 is a diagram showing the effect of DHCL, a composition of the present invention, on the activity of Nrf2 and its target genes, caveolin-1 and production of free radicals in RANKL treated osteoclast progenitor cells (BMMs):

A: effect of DHCL on mRNA expression of Nrf2 and target genes;

B: effect of DHCL on expression of target proteins including Nrf2 and antioxidant enzymes;

C: effect of DHCL on free radical accumulation in cells; And

D: Effect of DHCL on expression of caveolin-1.

FIG. 25 is a diagram showing the effects of DHCL at the time of exposure of the composition of the present invention on differentiation of RANKL treated osteoclast progenitor cells (BMMs) and expression of NFATc1 and its target genes and osteoclast fusion related genes:

A: Effects of DHCL on osteoclast differentiation at time of exposure; And

B: Effects of DHCL upon exposure to mRNA expression of NFATc1 and target genes.

FIG. 26 is a diagram showing the effects of DHCL at the time of exposure of the composition of the present invention on actining formation of RANKL-treated osteoclast progenitor cells (BMMs) and bone resorption on dentine discs:

A: Chart showing the experimental design of the exposure time point of DHCL;

B: Effect of time point of exposure of DHCL on the formation of actining; And

C: Effect of DHCL upon bone exposure on bone absorption.

27 is a diagram showing the inhibitory effect of DHCL, a composition of the present invention, on bone damage caused by LPS-induced inflammation:

A: imaging of TRAP stained skull;

B: Imaging of skull sections stained with TRAP and Hematoxilin; And

C: Analysis of the number of bone cavity and osteoclast of B.

28 is a diagram showing the concentration and time-dependent effect of cinconin (CN), the composition of the present invention on the differentiation of progenitor cells (BMMs) of RANKL-treated osteoclasts:

A: TRAP staining of osteoclasts differentiated by RANKL in the presence of various concentrations of CN;

B: analysis of the number of TRAP stained multinucleated osteoclasts of A;

C: concentration-specific cytotoxicity analysis 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 the number of TRAP stained multinucleated osteoclasts of D above; And

F: hourly cytotoxicity analysis of CN (Veh: Vehicle).

29 is a diagram showing the effect of cinconin (CN), the composition of the present invention on the expression of NFATc1 and target genes in RANKL treated osteoclasts (BMMs):

A: concentration-dependent effect of CN on the expression of NFATc1;

B: hourly effect of CN on expression of NFATc1; And

C: Effect of CN on mRNA expression of NFATc1 and target genes.

30 is a diagram showing the effect of cinconin (CN), a composition of the present invention, on NF-κB activity and c-FOS expression in progenitor cells (BMMs) of RANKL treated osteoclasts:

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 expression of c-Fos; And

D: Effect of CN on mRNA expression of c-Fos and NF-κB target genes.

Figure 31 shows the effect of cinconin (CN), a composition of the present invention, on phosphorylation of MAPKs and Src-AKT and FOXO1 in RANKL treated osteoclasts (BMMs):

A: effect of CN on phosphorylation of MAPKs;

B: effect of CN on phosphorylation of Src-AKT and FOXO1;

C: effect of CN on FOXO1 and catalase expression; And

D: Effect of CN on mRNA expression of catalase.

32 is a diagram showing the effect of cinconin (CN), the composition of the present invention on the activity of CREB-PGC-1β in RANKL treated osteoclasts (BMMs):

A: effect of CN on mRNA expression of PGC-1β and target genes;

B: effect of CN on expression of PGC-1β; And

C: Effect of CN on phosphorylation of CREB.

Figure 33 shows the effect of the exposure time of the composition of the present invention, Shinkonin (CN) on osteoclast differentiation and expression of NFATc1 and PGC-1β and their target genes, and RANKL-treated osteoclast progenitor cells (BMMs) Fig. 1 shows the effects of the composition of the present invention, cinconin (CN) on actining formation and bone resorption in dentine discs.

A: Effect of CN on exposure to osteoclast differentiation;

B: effect of CN exposure time on mRNA expression of NFATc1 and target genes;

C: effect of CN exposure time on mRNA expression of PGC-1β and target genes;

D: chart showing experimental design of exposure time of CN;

E: effect by time of exposure of CN on the formation of actining; And

F: Effect of CN on exposure to bone absorption.

34 is a diagram showing the inhibitory effect of cinconin (CN), the composition of the present invention on bone damage caused by LPS-induced inflammation:

A: Photograph of skull sections stained with TRAP and Hematoxilin; And

B: Analysis of the number of bone cavity and osteoclasts of A.

Hereinafter, the present invention will be described in detail.

Euphorbia Factor L1 (hereinafter referred to as EFL1), which is an active ingredient of the present invention, is a diterpenoid obtained from seeds of Euphorbia lathyris linne (Successor / Platinum). Has:

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

In addition, it is preferable that the Euphorbia factor L1 of this invention is 0.1-20 micrometers concentration. Euphorbia factor L1 composition having a concentration of less than 0.1 μM has a problem in that the treatment efficiency of bone disease is lowered, and if 20 μM or more, there is a problem that toxicity may occur in cells. In this respect, the concentration of the pharmaceutical composition containing Euphoria factor L1 as an active ingredient may be preferably 1 to 10 μM, more preferably 6 μM, but is not limited thereto.

The composition is characterized by reducing the expression or activity of NFATc1, NF-κB, c-FOS and PGC-1β, characterized by increasing the expression or activity of Nrf2, inhibits the production of free radicals in the cell It promotes the death of osteoclasts and is characterized by inhibiting bone damage caused by inflammation.

In addition, bone diseases to which the composition of the present invention can be applied include growth stage of development, fracture, osteoporosis due to excessive absorption of osteoclasts (osteoporosis), rheumatoid arthritis, periodontal disease (periodontal disease), Paget It is preferably at least one selected from the group consisting of Paget disease and metastatic bone cancers. It is not limited to this.

In a specific experimental example of the present invention, it was confirmed that the Euphorbia factor L1 (hereinafter referred to as EFL1) described in Chemical Formula 1 effectively inhibits osteoclast differentiation by RANKL (see FIG. 1). In addition, when EFL1 was treated, mRNA expression of NFATc1 protein, which is the most important transcription factor in osteoclast differentiation, and its target genes, CK, MMP9 and TRAP genes, was inhibited (see FIG. 2).

In addition, it was confirmed that phosphorylation of IκBα was suppressed in the presence of EFL1, NF-κB activity known as an upstream regulator of NFATc1, expression of its target gene SOD2, and c-Fos expression were also significantly inhibited (see FIG. 3). .

In addition, it was confirmed that EFL1 inhibited the mRNA expression of PGC-1β and its target genes, ND4, COX1, and COX3 (see FIG. 5), and mRNA expression of Nrf2 and NQO1 and Srx, which are representative target genes, increased together with EFL1. It confirmed that it became. At the same time, the expression of Nrf2 target proteins Srx, Prxl, PrxV, PrxVI, Trx1 and TrxR1 antioxidant enzymes, including Nrf2 and antioxidant enzymes, also increased. In addition, it was confirmed that the generation of free radicals is reduced by EFL1 (see Fig. 6).

In addition, it was confirmed that EFL1 inhibited maintenance of osteoclasts differentiated, actin ring formation, and bone resorption in the dentine disc (see FIGS. 7 and 8), and promoted the death of osteoclasts (see FIG. 9). It was confirmed that the bone damage by inhibiting (see Fig. 10).

Therefore, the composition containing the euphoria factor L1 of the present invention as an active ingredient can be usefully used for the prevention or treatment of bone diseases.

In addition, the scullcapflavone 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 (2):

Wherein R ₁ and R ₂ are H or OCH ₃ .

The present invention provides a pharmaceutical composition for preventing or treating bone diseases containing the compound represented by Formula 2, or a pharmaceutically acceptable salt thereof as an active ingredient.

The skull cap flavone derivatives of the present invention is characterized in that the skull cap flavone I or skull cap flavone II, wherein the skull cap flavone derivatives R ₁ and R ₂ are each independently hydrogen (H) or methoxy (OCH ₃ ) group.

Skullcapflavone I of the present invention, in the above formula (3) R ₁ and R ₂ is hydrogen (H).

Also, in the present invention, Scapcapflavone II, R ₁ and R ₂ in the formula (4) is a methoxy (OCH ₃ ) group.

In addition, the skullcapflavone derivative of the present invention is preferably in a concentration of 0.1 to 10 μM. Skullcapflavone derivative composition having a concentration of less than 0.1 μM has a problem that the treatment efficiency of bone disease is lowered, there is a problem that can cause toxicity to cells if more than 10 μM. In this aspect, the concentration of the pharmaceutical composition containing the scullcapflavone derivative as an active ingredient may be preferably 1 to 5 μM, more preferably 2 μM, but is not limited thereto.

In addition, the composition is characterized by reducing the expression or activity of NFATc1, c-FOS, MAPKS, Src, AKT, CREB, PGC-1β and caveolin-1, and increases the expression or activity of Nrf2 and FOXO1 and catalase Characterized in that it inhibits the production of free radicals in the cell and inhibits bone damage caused by inflammation.

In addition, bone diseases to which the composition of the present invention can be applied include growth stage of development, fracture, osteoporosis due to excessive absorption of osteoclasts (osteoporosis), rheumatoid arthritis, periodontal disease (periodontal disease), Paget It is preferably one or more selected from the group consisting of paget disease and metastatic bone cancers, but not limited thereto.

In a specific experimental example of the present invention, it was confirmed that the skull capflavone Ⅱ (hereinafter SFII) of the present invention, the skull cap flavone derivative described in Formula 2 effectively inhibits the differentiation of osteoclast progenitor cells (BMMs) by RANKL (See FIG. 11). In addition, mRNA treatment of CR and DC-STAMP genes was inhibited with SFII, along with NFATc1 protein, the most important transcription factor in osteoclast differentiation, and its target genes CK, MMP9, and TRAP (see FIG. 12). ).

In addition, it was confirmed that the expression of c-Fos protein and mRNA known as upstream regulator of NFATc1 was significantly suppressed in the presence of SFII (see FIG. 13), and that phosphorylation of MAPKS and Src-AKT and FOXO1 was inhibited ( See FIG. 14). As a result, the degradation of FOXO1 was also inhibited. As a result, the transcriptional activity of FOXO1 was increased, indicating that the expression of catalase, a target gene, was increased at both protein and mRNA levels (see FIG. 14).

In addition, SFII was found to significantly inhibit the mRNA expression of CREB and PGC-1β and its target genes ND4, COX1, COX3 and Cyt b (see FIG. 15). SFII and NQO1 and Srx are representative target genes by SFII. MRNA expression was increased together. Simultaneously, Nrf2 and Nrf2 target proteins including antioxidant enzymes, NQO1, Srx, PrxI, PrxII, PrxIII, PrxIV, PrxV, PrxVI, Trx1, Trx2, TrxR1 and TrxR2, were also found to increase expression. In addition, it was confirmed that the generation of reactive oxygen by SFII was reduced, and the expression of caveolin-1, which is known to inhibit Nrf2 activity and promote the differentiation of osteoclasts, was also inhibited by SFII (see FIG. 16).

In addition, SFII inhibits the formation of actin ring and bone resorption by osteoclasts in dentine disc (see FIGS. 17 and 18) and inhibits bone damage due to LPS inflammation (see FIG. 19).

Therefore, the composition containing the skull cap flavone derivative of the present invention as an active ingredient can be usefully used for the prevention or treatment of bone diseases.

In addition, the dihydrocostus lactone (DHCL) containing the present invention as an active ingredient has a structure represented by the following formula (5):

The present invention provides a pharmaceutical composition for preventing or treating bone diseases, comprising the compound represented by Formula 5, or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the dihydrocostus lactone of the present invention is characterized in that the concentration of 0.1 to 10 μM. Dihydrocostus lactone composition having a concentration of less than 0.1 μM has a problem that the treatment efficiency of bone disease is lowered, there is a problem that can cause toxicity to cells if more than 10 μM. In this aspect, the concentration of the pharmaceutical composition containing dihydrocostus lactone as an active ingredient may be preferably 1 to 5 μM, more preferably 1.5 μM.

In addition, the composition is characterized by reducing the expression or activity of NFATc1, c-FOS, JNK, ERK, Src, AKT and caveolin-1, increases the phosphorylation of CREB and the expression of PGC-1β, Nrf2 and FOXO1 And increasing expression or activity of catalase, and inhibiting the production of free radicals in cells and inhibiting bone damage caused by inflammation.

In addition, bone diseases to which the composition of the present invention can be applied include growth stage of development, fracture, osteoporosis due to excessive absorption of osteoclasts (osteoporosis), rheumatoid arthritis, periodontal disease (periodontal disease), Paget It is characterized in that any one or more selected from the group consisting of (Paget disease) and metastatic bone cancer (metastatic bone cancers).

In a specific experimental example of the present invention, it was confirmed that dehydrocostus lactone (DHCL) described in Chemical Formula 5 effectively inhibits the differentiation of osteoclast progenitor cells (BMMs) by RANKL (see FIG. 20). ). In addition, when treated with DHCL, mRNA expression of CR and DC-STAMP genes was suppressed along with NFATc1 protein, the most important transcription factor in osteoclast differentiation, and its target genes CK, MMP9, and TRAP (see FIG. 21). ).

In addition, it was confirmed that the expression of c-Fos protein and mRNA known as upstream regulator of NFATc1 was significantly inhibited in the presence of DHCL (see FIG. 22), and that phosphorylation of JNK, ERK, Src-AKT and FOXO1 was inhibited. (See FIG. 23). As a result, the degradation of FOXO1 was also inhibited. As a result, the transcriptional activity of FOXO1 was increased, indicating that the expression of catalase, a target gene, was increased at both protein and mRNA levels (see FIG. 23).

In addition, it was confirmed that mRNA expression of Nrf2 and NQO1 and Srx, which are representative target genes, was increased by DHCL, and Nrf2 target proteins including Nrf2 and antioxidant enzymes were also increased. In addition, it was confirmed that the generation of free radicals is reduced by DHCL, it was confirmed that the expression of caveolin-1, which inhibits the activity of Nrf2 and is known as a differentiation promoter of osteoclasts, is also inhibited by DHCL (see FIG. 24).

In addition, it was confirmed that DHCL inhibits the formation of actin ring and bone uptake by osteoclasts in dentine disc (see FIGS. 25 and 26) and inhibits bone damage due to LPS inflammation (see FIG. 27).

Therefore, the composition containing the dihydrocostus lactone (DHCL) of the present invention as an active ingredient can be usefully used for the prevention or treatment of bone diseases.

In addition, cinchonine (CN), which is contained as an active ingredient of the present invention, is an alkaloid derived from Cinchona bark, and has a structure represented by Formula 6 below:

The present invention provides a pharmaceutical composition for preventing or treating bone diseases, which contains the compound represented by Chemical Formula 6, or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the cinconin of the present invention is characterized in that the concentration of 0.1 to 50 μM. Synconin composition having a concentration of less than 0.1 μM has a problem in that the treatment efficiency of bone disease is lowered, there is a problem that can cause toxicity to cells if more than 50 μM. In this aspect, it may be preferable that the concentration of the pharmaceutical composition containing cinnaconin as an active ingredient is 1 to 30 μM, more preferably 20 μM.

The composition is characterized by reducing the expression or activity of NFATc1, NF-κB, ERK, Src, AKT, FOXO1, c-FOS and PGC-1β, it is characterized by inhibiting bone damage caused by inflammation.

In addition, bone diseases to which the composition of the present invention can be applied include growth stage of development, fracture, osteoporosis due to excessive absorption of osteoclasts (osteoporosis), rheumatoid arthritis, periodontal disease (periodontal disease), Paget It is preferably one or more selected from the group consisting of paget disease and metastatic bone cancers, but not limited thereto.

In a specific experimental example of the present invention, it was confirmed that the cinconin (CN) of the present invention described in the formula (6) effectively inhibits the differentiation of osteoclasts by RANKL (see Fig. 28). In addition, NFATc1 protein, which is the most important transcription factor in osteoclast differentiation, and its target genes, CK, MMP9 and TRAP genes, were inhibited when Cynconin was treated (see FIG. 29).

In addition, phosphorylation of IκBα was inhibited in the presence of Cinconin, and expression of NF-κB and its target genes SOD2 and COX2, which are known as upstream regulators of NFATc1, was also inhibited. In addition, it was confirmed that c-Fos expression was significantly inhibited by cinnaconin (see FIG. 30), and phosphorylation of ERK, Src-AKT, and FOXO1 was inhibited (see FIG. 31).

In addition, it was confirmed that synonin inhibited the mRNA expression of PGC-1β and its target genes, ND4, Cytb, COX1, COX3 (see FIG. 32), and synonin induced by osteoclasts in the formation of actin ring and dentine disc. It was confirmed that inhibition of bone absorption (see FIG. 33), and inhibiting bone damage caused by LPS inflammation (see FIG. 34).

Therefore, the composition containing the present mykonin (CN) as an active ingredient can be usefully used for the prevention or treatment of bone diseases.

In addition, the present invention includes not only the compound represented by Chemical Formula 1 to Chemical Formula 6, but also a pharmaceutically acceptable salt thereof, and possible solvates, hydrates, racemates or stereoisomers which can be prepared therefrom.

The present invention can be used in the form of a compound represented by Formula 1 to Formula 6 or a pharmaceutically acceptable salt thereof, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt. . Acid addition salts include 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, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as doates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide and iodide Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfo Nate, Xylene Sulfonate, Phenyl Acetate, Phenylpropionate, Phenyl Butyrate, Citrate, Lactate, Hydroxybutyrate, Glycolate, Maleate, Tartrate, Methanesulfonate, Propanesulfonate, Naphthalene-1-sulfonate , Naphthalene-2-sulfonate or mandelate.

Acid addition salts according to the present invention are dissolved in conventional methods, for example, the compounds represented by the above formulas (1) to (6) in an excess of aqueous acid solution, and the salts are water-miscible organic solvents such as methanol, ethanol, acetone. Or by precipitation with acetonitrile. In addition, the same amount of the compound represented by the formula (1) to formula (6), and the acid aqueous solution or alcohol may be heated, and then the mixture is evaporated to dryness or the precipitated salt may be produced by suction filtration.

Bases can also be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess 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 pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg, silver nitrate).

When formulating the composition, it is prepared using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants.

Solids for oral administration include tablets, pills, powders, granules, capsules, troches and the like, and such solid preparations include at least one excipient example in the compound represented by Formula 1 to Formula 6 of the present invention. For example, it is prepared by mixing starch, calcium carbonate, sucrose or lactose or gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions, or syrups, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like.

As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 81, cacao butter, taurine, glycerol, gelatin and the like can be used.

The composition according to the 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 an effective dose level means the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum 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 weight of the patient, and in general, 0.1 mg to 100 mg, preferably 0.5 mg to 10 mg per 1 kg of body weight is administered daily or every other day. Alternatively, the administration may be divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

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

In another aspect, the present invention provides a dietary supplement for the improvement of bone disease containing Euphorbia factor L1 represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food for the improvement of bone disease containing the skull cap flavone derivative represented by the formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient. In addition, the skull cap flavone derivative is characterized in that the skull cap flavone I or skull cap flavone II.

In another aspect, the present invention provides a dietary supplement for improving bone diseases containing dihydrocostus lactone represented by the formula (5) 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 the compound represented by the formula (6), or a pharmaceutically acceptable salt thereof as an active ingredient.

The bone diseases include growth stage development, fracture, osteoporosis (osteoporosis) due to excessive bone resorption of osteoclasts (rheumatoid arthritis), periodontal disease (periodontal disease), Paget disease (metastatic) and metastatic bone cancer (metastatic) It is preferably at least one selected from the group consisting of bone cancers), but is not limited thereto.

The "health functional food" of the present specification is manufactured by using nutrients or ingredients (functional raw materials) having useful functions to the human body, which are easily deficient in a daily meal, and maintaining health through physiological functions or maintaining normal functions of the human body. The food is to maintain and improve the food as defined by the Commissioner of Food and Drug Safety, but is not limited to this and is not used to exclude the health food in the normal sense.

The composition of the present invention may be added as it is to food or used with other food or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). In general, the amount of the compound in the dietary supplement may be added at 0.01 to 90 parts by weight of the total food weight. However, in the case of prolonged ingestion for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the composition as an essential ingredient in the indicated ratios, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general 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 conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tautin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)} and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. The proportion of said natural carbohydrates 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, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages.

These components can be used independently or in combination. The proportion of such additives is not so critical 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.

The present invention also provides a method for preventing or treating bone diseases, comprising administering Euphorbia Factor L1 or a pharmaceutically acceptable salt thereof to a mammal, which is represented by Formula 1 below:

[Formula 1]

The present invention also provides the use of Euphorbia Factor L1 or a pharmaceutically acceptable salt thereof, as described in Formula 1, for use in the manufacture of a medicament for the prevention or treatment of bone diseases.

The present invention also provides a method for preventing or treating bone diseases, comprising administering to a mammal a skullcapflavone derivative represented by Formula 2 or a pharmaceutically acceptable salt thereof:

[Formula 2]

Wherein R ₁ and R ₂ are H or OCH ₃ .

The present invention also provides the use of a scapcapflavone derivative according to Formula 2 or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for the prevention or treatment of bone diseases.

In addition, the present invention provides a method for preventing or treating bone diseases, comprising administering to a mammal a dihydrocostus lactone represented by the following formula (5) or a pharmaceutically acceptable salt thereof:

[Formula 5]

The present invention also provides the use of a dihydrocostus lactone or a pharmaceutically acceptable salt thereof according to Formula 5, for use in the manufacture of a medicament for the prevention or treatment of a bone disease:

In another aspect, the present invention provides a method for preventing or treating bone diseases, comprising administering to a mammal, a neoconsin or a pharmaceutically acceptable salt thereof represented by the following formula:

[Formula 6]

The present invention also provides the use of cinconin or a pharmaceutically acceptable salt thereof as described in Formula 6 for use in the manufacture of a medicament for the prevention or treatment of bone diseases.

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

However, the following experimental examples are only illustrative of the present invention, and the content of the present invention is not limited by the following experimental examples.

I. Of EFL1  Efficacy Check

< Experimental Example  1> Of EFL1  Osteoclast differentiation inhibition

Euphorbia factor L1 (hereinafter referred to as EFL1) contained in the present invention as an active ingredient was used in an experiment using CFN92883 / 76376-43-7 manufactured by ChemFaces.

First, in order to confirm the osteoclast differentiation inhibitory effect of EFL1, experiments were performed on BMM (Bone Marrow-Derived Macrophages) cells. BMM cells are precursor cells capable of differentiating into osteoclasts. In this experiment, cells extracted from femur and tibia bone marrow of 8-week-old C57BL / 6 male mice were used.

 BMM (Bone Marrow-Derived Macrophages) cells were treated with 50 ng / ml RANKL for 3 days in the presence of various concentrations (0, 4, 6, 8 μM) of EFL1 to differentiate into osteoclasts and 4% paraformaldehyde after PBS washing. After fixation with TRAP staining using leukocyte acid phosphatase cytochemistry kit (Sigma Aldrich), the number of osteoclasts with three or more multinucleated stained TRAP was analyzed under a microscope.

As a result, osteoclast differentiation was effectively suppressed from 6 μM of EFL1 (A and B of FIG. 1).

In addition, the cytotoxicity was analyzed by EASY Cytox (WST-1) assay kit in order to exclude that the osteoclast differentiation inhibitory effect of EFL1 is due to cytotoxicity. 10 μl of 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium monosodium salt (WST-1) reagent (Roche Applied Science) After incubation at 37 ° C. for 2-4 hours, absorbance at 450 nm was measured.

As a result, it was confirmed that cytotoxicity by EFL1 did not appear up to 8 μM concentration (FIG. 1C). In addition, osteoclast differentiation was significantly inhibited even when BMM cells were differentiated by treatment of RANKL for 3 to 5 days in the presence of 6 μM EFL1 (D and E in FIG. 1), and no cytotoxicity was observed until day 5 It was confirmed (FIG. 1 F). This shows that the effect of inhibiting osteoclast differentiation of EFL1 is not due to apoptosis.

< Experimental Example  2> On EFL1  by NFATc1  Activation Suppression

In order to confirm the osteoclast differentiation inhibitory effect of EFL1, BMM cells were treated with 50 ng / ml of RANKL for 2 days in the presence of various concentrations (0, 4, 6, 8 μM) of EFL1 to differentiate into osteoclasts, and osteoclasts. In order to confirm the expression of NFATc1 protein, the most important transcription factor in cell 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 nonspecific binding sites on the membrane were blocked using 5% skim milk dissolved in TBST (0.05% Tween-20 in Tris-buffered saline, pH 7.4). Primary antibodies (mouse monoclonal antibodies, Santa Cruz Biotechnology Inc) were treated at 4 ° C. for 14 hours and secondary antibodies were treated at room temperature for 1 hour and analyzed by West save Up (Ab Frontier).

As a result, NFATc1 protein was significantly inhibited from 4 μM EFL1 concentration (FIG. 2A), and it was confirmed that the inhibitory effect was continued up to 5 days (FIG. 2B).

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

For real time-PCR, total RNA was extracted using Trizol reagent (Invitrogen) and quantified at absorbance at 260 nm. Total RNA was totaled to 15 ml, containing 0.5 mg of oligo (dT) primers. After 5 minutes incubation at ℃, it was cooled directly on ice. The first strand of cDNA was synthesized at 25 μl for 1 hour at 42 ° C. and 10 minutes at 70 ° C. in 25 μl of final volume containing M-MLV reverse transcriptase (Promega) 200 units, ribonuclease inhibitor 24 units and 0.25 mM each of dNTP. ABI 7300 real at a final 20 μl volume containing 10 μl of 2 × SYBR Green PCR Master Mix (M Biotech) and 1 μl of 10 μμM of each gene-specific primer (Genotech). Amplification was carried out using a time PCR system (Applied Biosystems), and the amplification was performed by performing 40 cycles for 2 minutes at 50 ° C, 2 minutes at 95 ° C, then 15 seconds at 95 ° C, and 1 minute at 60 ° C. The sequence of Primer used in the experiment is 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-1β	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
CK, cathepsin K; MMP9, matrix metalloprotease 9; SOD2, superoxide dismutase 2; COX2, cyclooxygenase 2

As a result, when treated with 50 ng / ml RANKL for 2 days in the presence of 6 μM EFL1, it was confirmed that the mRNA of NFATc1 and its target genes CK, MMP9 and TRAP genes are also significantly suppressed (FIG. 2C). .

< Experimental Example  3> On EFL1  by NF - κB  And c- FOS  control

To determine whether EFL1 modulates NF-κB activity and c-Fos expression, known as upstream regulators of NFATc1, immunoblot using IκBα and p-IκBα (rabbit polyclonal antibodies, Cell Signaling Technology) was used to determine the degree of phosphorylation of IκBα. SOD2 and c-FOS protein expression was also confirmed by immunoblot using antibodies (SOD2 / rabbit polyclonal antibody, Upstate; c-FOS / rabbit polyclonal antibody, Santa Cruz Biotechnology, Inc), and SOD2 and c-Fos mRNA expression was confirmed. It was analyzed by real-time PCR by the method described in Experimental Example 2 (see Table 1 primer sequence).

As a result, EFL1 (6 μM) significantly inhibited the phosphorylation of IκBα in the presence of RANKL (50 ng / ml) (FIG. 3A) and significantly inhibited protein and mRNA expression of SOD2, an NF-κB target gene. (B and D of FIG. 3). In addition, EFL1 significantly inhibited c-Fos protein and mRNA expression (Fig. 3 C and D).

< Experimental Example  4> On EFL1  by MAPKs  And AKT  Active inhibition

To see the effects of EFL1 on MAPKs and AKT activated by RANKL, rabbit polyclonal antibodies to phospho-JNK, phospho-ERK, phospho-p38, phospho-AKT, rabbit monoclonal antibody to AKT, Cell Signaling Technology: Comparison of the phosphorylation levels of MAPKs and AKT proteins by immunoblot using Rabbit polyclonal antibodies against ENK1, p38, ERK2 mouse monoclonal antibody, Santa Cruz Biotechnology, Inc. It was confirmed that it is not affected by (Fig. 4).

< Experimental Example  5> On EFL1  by PGC - 1β  Active inhibition

PGC-1β is a transcription factor well known to be activated by RANKL and involved in osteoclast differentiation. In order to confirm the PGC-1β regulatory effect of EFL1, mRNA expression of PGC-1β and its target genes ND4, COX1 and COX3 was confirmed by real-time PCR (see Table 1 primer sequence).

As a result, EFL1 (6 μM) significantly inhibited PGC-1β, ND4, COX1 and COX3 mRNA expression by RANKL (50 ng / ml) (FIG. 5A). In addition, EFL1 also inhibited the expression of PGC-1β (rabbit polyclonal antibody, Abcam) protein, but CREB (phospho-CREB rabbit monoclonal antibody, Cell Signaling Technology; CREB mouse monoclonal antibody, Santa Cruz Biotechnology) , Inc) did not affect the phosphorylation (B and C of Figure 5).

< Experimental Example  6> On EFL1  by Nrf2  Activation

Redox-sensitive transcription factor Nrf2 is known to be involved in osteoclast differentiation by regulating the expression of GSH synthase and antioxidant enzymes in vivo, and to investigate the inhibitory effect of osteoclast differentiation by regulating Nrf2 activity of EFL1. , Nrf2, NQO1 and Srx gene expression was confirmed by real-time PCR (see Table 1 primer sequence), Nrf2 target proteins NQO1, Srx, Prxl, PrxV, PrxVI, Trx1 and TrxR1 (rabbit polyclonal antibodies, Young In Frontier) protein expression was confirmed by immunoblot.

As a result, mRNA expression of Nrf2 and its target genes NQO1 and Srx was increased by EFL1 (6 μM) treatment in the presence of RANKL (50 ng / ml) (FIG. 6A). In addition, Nrf2 and its target proteins NQO1, Srx, Prxl, PrxV, PrxVI, Trx1 and TrxR1 antioxidant enzymes were also confirmed to increase the expression (Fig. 6B).

In addition, it is well known that free radicals are produced and involved in the differentiation process of osteoclasts by RANKL. If EFL1 activates Nrf2 and the expression of Nrf2 target proteins including antioxidant enzymes is increased, these antioxidant enzymes may be involved in RANKL. In order to confirm this possibility, the active oxygen generated by the reaction may be removed, and RANKL was treated for 2 days in the presence of EFL1, and reactive oxygen species (ROS) were measured.

ROS was measured by oxidant sensitive fluorescence detection using CM-H2DCFDA. Since CM-H2DCFDA reacts with ROS and fluoresces, cells are treated with RANKL, washed with HBSS, and then treated with 5 μM of CM-H2DCFDA for 10 minutes in the dark, using a FACS Calibur flow cytometer (BD Biosciences). Analyzed.

As a result, it was confirmed that free radicals in the cells were significantly reduced in the presence of EFL1 (FIG. 6A, lower left).

In addition, EFL1 (6 μM) was pretreated for 30 minutes and RANKL (A in FIG. 6, bottom) or H ₂ O ₂ (FIG. A, lower 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 free radicals in cells were reduced by EFL1 (A, middle and lower right in FIG. 6). Since EFL1 was pretreated only for a short time before the expression of antioxidant enzymes by Nrf2 activation, this result indicates that free radicals can be directly removed by EFL1.

< Experimental Example  7> On EFL1  Inhibition of bone resorption by osteoclasts

In order to investigate the differentiation inhibitory effect of EFL1 osteoclasts, different time points (E0, RANKL treatment; E1, RANKL 1 day treatment; E2, RANKL 2 days) during RANKL treatment to differentiate BMM cells into osteoclasts Post-treatment) was exposed to EFL1 and analyzed for the number of TRAP stained multinucleated osteoclasts.

As a result, when EFL1 (6 μM) was treated with RANKL (50 ng / ml) (E0), the formation of osteoclasts was significantly inhibited even when EFL1 was treated (E2) 2 days later (Fig. 7). A). Expression of NFATc1 and its target genes CK, MMP9, and TRAP under the same experimental conditions was also measured by real-time PCR (see Table 1 primer sequence). After 2 days of treatment with RANKL, RANKL and It was significantly inhibited as when treated together (FIG. 7B). And it was confirmed that the expression of PGC-1β and its target genes ND4, COX1, COX3, Cyt b was also significantly inhibited by EFL1 exposed 2 days after treatment with RANKL (Fig. 7 C).

In addition, in order to see the effect of EFL1 on the formation of acting ring and bone resorption under similar experimental conditions, BMM cells were treated with RANKL for 3 days to differentiate into osteoclasts, and then treated with EFL1, and EFL1 The cells treated with RANKL were fixed as follows and stained with actining and nuclei or analyzed for bone resorption on dentine discs. The cells were fixed in PBS with 3.7% formaldehyde and treated with 0.1% Triton X-100, followed by Alexa Fluor 488-phalloidin (Invitrogen) for 20 minutes, followed by DAPI (4 ', 6-diamidino-2-phenylindole). , Roche) and observed through a fluorescence microscope. In addition, the degree of bone resorption was treated with BMM cells cultured in dentine disc (Immunodiagnostic Systems Ltd) under the same conditions as above, followed by removal of the cells using a cotton tip, staining the resorption pit with hematoxylin, and then using a microscope 100x magnification. The photographs taken were analyzed with Image-Pro Plus 4.5 software (Media Cybernetics).

 As a result, it was confirmed that EFL1 completely inhibited the formation of acting rings and bone resorption by osteoclasts in the dentine disc in both cases (A, B and C of FIG. 8). These results show that EFL1 can inhibit osteoclast formation and bone resorption.

< Experimental Example  8> On EFL1  Death of osteoclasts

The differentiation of BMM cells into osteoclasts and the inhibition of osteoclasts, actin ring formation, and bone resorption by EFL1 after treatment induced differentiation of osteoclasts differentiated from EFL1. The osteoclasts were treated with EFL1 (6 μM) and the shape change of cells with time (0, 6, 12, 18h) was observed through a microscope.

As a result, the osteoclasts treated only with the vehicle became larger due to the fusion of the cells, whereas the osteoclasts treated with the EFL1 disappeared over time (Fig. 9A). In addition, as a result of measuring cell growth through the WST-1 assay, it was confirmed that EFL1 inhibits the growth of cells (B of FIG. 9). These results show that EFL1 does not affect the growth of BMM cells but inhibits the growth of differentiated osteoclasts as shown in Experimental Example 1.

In addition, to confirm the induction of differentiated osteoclasts under the same conditions, the osteoclasts were fixed as follows, followed by DAPI staining and TUNEL assay: The osteoclasts were fixed with 4% formaldehyde for 60 minutes at room temperature and washed with PBS. After staining the cells in which apoptosis occurred using In Situ Cell Death Detection Kit TMR red (Roche), the cells were stained with DAPI for 3 minutes at room temperature, and observed with a fluorescence microscope. As a result, it was confirmed that EFL1 induces the death of differentiated osteoclasts (C and D of FIG. 9).

< Experimental Example  9> Of EFL1  Suppress bone damage caused by inflammation

In order to confirm the inhibitory effect of bone damage caused by inflammation of EFL1 in an animal model, LPS (Lipopolysaccharide, 12.5 mg / kg body weight), an inflammatory inducer, was injected into the mouse skull twice daily at intervals. At this time, vehicle (40% DMAC + 10% Tween 80 + 50% distilled water) or EFL1 (10 mg / kg) was administered together, and 5 days after the first injection, the skull was fixed with 4% paraformaldehyde and PBS washed. In addition, decalcification was performed with 0.5 M ethylenediaminetetraacetic acid for 7 days, paraffin block was made and then cut and stained with TRAP and Hematoxylin.

As a result, there were many TRAP stained parts in the skull treated only with LPS, but the TRAP stained part was significantly reduced in the skull treated with EFL1 (FIG. 10A). In addition, as a result of quantitative analysis, it was confirmed that the number of bone cavity and osteoclasts increased by LPS treatment was significantly reduced by EFL1 (FIG. 10B).

II. SF Confirmation of efficacy of Ⅱ

< Experimental Example  10> SF Suppression of osteoclast differentiation of Ⅱ

Skullcapflavone II (hereinafter referred to as SFII) containing the present invention as an active ingredient was used in an experiment using CFN92216 / 55084-08-7 manufactured by ChemFaces.

First, in order to confirm the osteoclast differentiation inhibitory effect of SFII, the present inventors performed experiments on BMM (Bone Marrow-Derived Macrophages) cells. BMM cells are precursor cells capable of differentiating into osteoclasts. In this experiment, cells extracted from femur and tibia bone marrow of 8-week-old C57BL / 6 male mice were used.

Bone Marrow-Derived Macrophages (BMM) cells were treated with 25 ng / ml M-CSF (macrophage colony stimulating factor) and 50 ng / ml RANKL in the presence of SFII at various concentrations (0, 1, 2, 3 μM). After treatment for 1 day, differentiated into osteoclasts, fixed with 4% paraformaldehyde after PBS wash, followed by TRAP staining using leukocyte acid phosphatase cytochemistry kit (SigmaAldrich), followed by TRAP staining. Analyzed.

As a result, osteoclast differentiation was effectively suppressed from SFII of 2 μM (FIGS. 11A and 11B).

In addition, cytotoxicity was analyzed by EASY Cytox (WST-1) assay kit in order to exclude that the osteoclast differentiation inhibitory effect of SFII is due to cytotoxicity. BMM cells were prepared using 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium mono-sodium salt (WST-1) reagent (Roche Applied Science), respectively. μl was added 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 by SFII did not appear up to a concentration of 3 μM (FIG. 11C). In addition, osteoclast differentiation was significantly inhibited even when BMM cells were differentiated by treatment with RANKL for 3 to 5 days in the presence of 2 μM SFII (D and E in FIG. 11), and no cytotoxicity was observed until day 5 It was confirmed (FIG. 11F). This shows that the osteoclast differentiation inhibitory effect of SFII is not due to apoptosis.

< Experimental Example  11> SF By Ⅱ NFATc1  Activation Suppression

In order to confirm the inhibitory effect of SFII on osteoclast differentiation, BMM cells were treated with 50 ng / ml of RANKL for 2 days in the presence of SFII at various concentrations (0, 1, 2, 3 μM), and differentiated into osteoclasts. In order to confirm the expression of NFATc1 protein, the most important transcription factor in cell 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 nonspecific binding sites on the membrane were blocked using 5% skim milk dissolved in TBST (0.05% Tween-20 in Tris-buffered saline, pH 7.4). Primary antibodies (mouse monoclonal antibodies, Santa Cruz Biotechnology Inc) were treated at 4 ° C. for 14 hours and secondary antibodies were treated at room temperature for 1 hour and analyzed by West save Up (Ab Frontier).

As a result, it was confirmed that NFATc1 protein was significantly suppressed from 2 μM SFII concentration (FIG. 2A), and the inhibitory effect was continued until 5 days (FIG. 12B).

In addition, mRNA expression changes of DC-STAMP genes associated with CK, MMP9 and TRAP genes, which are target genes of NFATc1, and CR and osteoclasts, which are calcitonin receptor genes, were confirmed by real time-PCR.

For real time-PCR, total RNA was extracted using Trizol reagent (Invitrogen) and quantified at absorbance at 260 nm. Total RNA was adjusted to 15 ml total containing 0.5 mg oligo (dT) primers. After 5 minutes incubation at ℃, it was cooled directly on ice. The first strand of cDNA was synthesized at 25 μl for 1 hour at 42 ° C. and 10 minutes at 70 ° C. at 25 μl of final volume containing M-MLV reverse transcriptase (Promega) 200 units, ribonuclease inhibitor 24 units and 0.25 mM each of dNTP. ABI 7300 real at the final 20 μl volume containing 0.8 μl of the diluted cDNA template (1: 2.5) containing 10 μl of 2X SYBR Green PCR Master Mix (M Biotech) and 1 μl of 10 μM of each gene-specific primer (Genotech). Amplification was carried out using a time PCR system (Applied Biosystems), and the amplification was performed by performing 40 cycles for 2 minutes at 50 ° C, 2 minutes at 95 ° C, then 15 seconds at 95 ° C, and 1 minute at 60 ° C. The sequence of Primer used in the experiment is shown in Table 2 below.

real-time PCR  the sequence of the primer and amplicon  size

Gene		Primer (sense & antisense)	SEQ ID NO:	Amplicon length (bp)
17	NFATc1	5'-CTTCAGCTGGAGGACACC-3 '	33	79
		5'-CCAATGAACAGCTGTAGCG-3 '	34
18	CK	5'-ACCACTGCCTTCCAATACG-3 '	35	99
		5’-CGTGGCGTTATACATACAAC-3 ’	36
19	MMP9	5’-AGACGACATAGACGGCATC-3 ’	37	97
		5’-TGCTGTCGGCTGTGGTTC-3 ’	38
20	TRAP	5'-CCAGCGACAAGAGGTTCC-3 '	39	113
		5'-AGAGACGTTGCCAAGGTGAT-3 '	40
21	SOD2	5'-ATTAACGCGCAGATCATGCA-3 '	41	161
		5'-TGTCCCCCACCATTGAACTT-3 '	42
22	COX2	5'-GATCATAAGCGAGGACCTG-3 '	43	85
		5'-GTCTGTCCAGAGTTTCACC-3 '	44
23	c-Fos	5'-GAGAAACGGAGAATCCGAAG-3 '	45	97
		5'-GAGAAACGGAGAATCCGAAG-3 '	46
24	PGC-1β	5'-CTCCAGGCAGGTTCAACCC-3 '	47	83
		5'-GGGCCAGAAGTTCCCTTAGG-3 '	48
25	ND4	5'-CATCACTCCTATTCTGCCTAGCAA-3 '	49	74
		5'-TCCTCGGGCCATGATTATAGTAC-3 '	50
26	COX1	5'-TTTTCAGGCTTCACCCTAGATGA-3 '	51	81
		5'-GAAGAATGTTATGTTTACTCCTACGAATATG-3 '	52
27	COX3	5'-CGGAAGTATTTTTCTTTGCAGGAT-3 '	53	82
		5'-CAGCAGCCTCCTAGATCATGTG-3 '	54
28	Cytb	5'-GCCACCTTGACCCGATTCT-3 '	55	64
		5'-TTGCTAGGGCCGCGATAAT-3 '	56
29	Nrf2	5'-TCTCCTCGCTGGAAAAAGAA-3 '	57	498
		5'-AATGTGCTGGCTGTGCTTTA-3 '	58
30	NQO1	5'-TTCTCTGGCCGATTCAGAG-3 '	59	180
		5'-GGCTGCTTGGAGCAAAATAG-3 '	60
31	Srx	5'-AGCCTGGTGGACACGATC-3 '	61	97
		5'-AGGAATAGTAGTAGTCGCCA-3 '	62
32	b-actin	5'-ACCCTAAGGCCAACCGTG-3 '	63	81
		5'-GCCTGGATGGCTACGTAC-3 '	64
33	CR	5'-TGATGACTCTCAGGACAATG-3 '	65	87
		5'-ACTGGATCAATCTGTAGGAG-3 '	66
34	DC-STAMP	5'-TTATGTGTTTCCACGAAGCCCTA-3 '	67	141
		5'-ACAGAAGAGAGCAGGGCAACG-3 '	68
35	Catalase	5'-CTCGTTCAGGATGTGGTTTTC-3 '	69	145
		5'-CTTTCCCTTGGAGTATCTGGTG-3 '	70
CK, cathepsin K; MMP9, matrix metalloprotease 9; SOD2, superoxide dismutase 2; COX2, cyclooxygenase 2

As a result, when 50 ng / ml RANKL was treated for 2 days in the presence of 2 μM SFII, the expression of CR and DC-STAMP genes, together with NFATc1 and its target genes CK, MMP9 and TRAP, was also significantly suppressed. (C of FIG. 12).

< Experimental Example  12> SF C-by II FOS  control

To determine whether SFII regulates NF-κB activity and c-Fos expression, known as upstream regulators of NFATc1, immunoblot using IκBα and p-IκBα (rabbit polyclonal antibodies, Cell Signaling Technology) was used to determine the degree of phosphorylation of IκBα. Expression of SOD2 and c-FOS proteins was also confirmed by immunoblot using these antibodies (SOD2 / rabbit polyclonal antibody, Upstate; c-FOS / rabbit polyclonal antibody, Santa Cruz Biotechnology, Inc), and SOD2 and c-Fos. Expression of mRNA was analyzed by real-time PCR by the method described in Experimental Example 11 (see Table 2 primer sequence).

As a result, SFII (2 μM) did not significantly affect the phosphorylation of IκBα in the presence of RANKL (50 ng / ml) (FIG. 13A), and also greatly influenced the protein and mRNA expression of SOD2, an NF-κB target gene. Not given (B and D in FIG. 13). However, SFII was found to significantly inhibit the expression of c-Fos protein and mRNA (C and D of Figure 13).

< Experimental Example  13> SF By Ⅱ MAPKs  And AKT  Active inhibition

To see the effect of SFII on MAPKs and AKT activated by RANKL, Rabbit polyclonal against their antibodies (phospho-JNK, phospho-ERK, phospho-p38, phospho-Src, phospho-AKT, phospho-Foxo1, Src) We compared the phosphorylation levels of MAPKs and AKT-related proteins by immunoblot using rabbit monoclonal antibodies against AKT, Foxo1, Cell Signaling Technology; Rabbit polyclonal antibodies against JNK1, p38, ERK2 mouse monoclonal antibody, Santa Cruz Biotechnology, Inc. saw.

As a result, it was confirmed that both phosphorylation of MAPKs and Src-AKT is inhibited by SFII (A and B in Fig. 14). In addition, AKT phosphorylates FOXO1, which induces degradation and inhibits migration to the nucleus, and further confirms the effect of SFII on phosphorylation of FOXO1. As a result, it was confirmed that the phosphorylation of FOXO1 was inhibited (FIG. 14B). . SFII inhibited the phosphorylation of FOXO1 by inhibiting AKT activity, thereby confirming that the degradation of FOXO1 was also inhibited (FIG. 14C). As a result, the transcriptional activity of FOXO1 was increased and the expression of catalase, a target gene, was increased at both protein and mRNA levels by immunoblot and real-time PCR (FIG. 14C and D).

< Experimental Example  14> SF By Ⅱ CREB  - PGC - 1β  Active inhibition

PGC-1β is a transcription factor well known to be activated by RANKL and involved in osteoclast differentiation. In order to confirm the PGC-1β regulatory effect of SFII, mRNA expression of PGC-1β and its target genes ND4, COX1, COX3 and Cyt b was confirmed by real-time PCR (see Table 2 primer sequence).

As a result, SFII (2 μM) significantly inhibited the mRNA expression of PGC-1β, ND4, COX1, COX3 and Cyt b by RANKL (50 ng / ml) (FIG. 15A). In addition, SFII also inhibited the expression of PGC-1β protein, CREB (phospho-CREB rabbit monoclonal antibody, Cell Signaling Technology; CREB mouse monoclonal antibody, Santa Cruz Biotechnology, a higher transcription factor of PGC-1β (rabbit polyclonal antibody, Abcam)) , Inc) also inhibited the phosphorylation (B and C of Figure 15). This indicates that SFII inhibits the CREB-PGC-1β pathway.

< Experimental Example  15> SF By Ⅱ Nrf2  Activation and free radical regulation

The redox-sensitive transcription factor Nrf2 is known to be involved in the differentiation of osteoclasts by regulating the expression of GSH synthase and antioxidant enzymes in vivo, and to investigate the inhibitory effect of osteoclast differentiation by regulating Nrf2 activity of SFII. , Nrf2, NQO1 and Srx gene expression was confirmed by real-time PCR (see Table 2 primer sequence), Nrf2 and other target proteins of Nrf2 including Nrf2 and antioxidant enzymes NQO1, Srx, PrxI, PrxII, PrxIII, PrxIV, PrxV, Expression of PrxVI, Trx1, Trx2, TrxR1 and TrxR2 (rabbit polyclonal antibodies, Young In Frontier) proteins 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 SFII (2 μM) treatment in the presence of RANKL (50 ng / ml) (FIG. 16A). In addition, Nrf2 and antioxidant enzymes, including NQO1, Srx, PrxI, PrxII, PrxIII, PrxIV, PrxV, PrxVI, Trx1, Trx2, TrxR1 and TrxR2 were also found to increase the expression (Fig. 16B).

In addition, it is well known that free radicals are produced and involved in the differentiation process of osteoclasts by RANKL. SFII activates Nrf2 to increase the expression of Nrf2 target proteins including antioxidant enzymes. In order to confirm this possibility, the active oxygen generated by the reaction may be removed, and RANKL was treated for 2 days in the presence of SFII and the reactive oxygen species (ROS) were measured.

ROS was measured by oxidant sensitive fluorescence detection using CM-H2DCFDA. Since CM-H2DCFDA reacts with ROS and fluoresces, cells are treated with RANKL, washed with HBSS, and then treated with 5 μM of CM-H2DCFDA for 10 minutes in the dark, using a FACS Calibur flow cytometer (BD Biosciences). Analyzed.

As a result, it was confirmed that the free radicals in the cell significantly decreased in the presence of SFII (C left side of Figure 16).

In addition, SFII (2 μM) was pretreated for 30 minutes and RANKL (C, M in Figure 16) or H ₂ O ₂ (C in Figure 16) to determine the possibility of SFII to remove the active oxygen directly as an antioxidant. , Right) was treated for 15 minutes and the active oxygen was measured using CM-H2DCFDA as described above.

As a result, it was confirmed that the free radicals in the cells were reduced by SFII (C, middle and right in Fig. 16). Since SFII was pretreated only for a short time before the expression of antioxidant enzyme by Nrf2 activation, this result means that free radicals can be directly removed by SFII.

In addition, since caveolin-1 regulates the expression of Nrf2 and is known as a facilitator of osteoclast differentiation, experiments with the effect of SFII on the expression of caveolin-1 showed that SFII is caveolin-1 (rabbit monoclonal) induced by RANKL. It was confirmed that the expression of antibody, Trnasduction Laboratories) is suppressed (FIG. 16D). This suggests that SFII inhibits the expression of caveolin-1, thereby reducing the binding of Nrf2, thereby promoting the activity of Nrf2 and consequently lowering free radicals in cells.

< Experimental Example  16> SF Inhibition of Bone Resorption of Osteoclasts by Ⅱ

In order to investigate the differentiation inhibitory effect of SFII osteoclasts, during differentiation of BMM cells into osteoclasts by treatment of RANKL, various time points (E0, RANKL treatment; E1, RANKL after 1 day treatment; E2, RANKL 2 days) After treatment) was exposed to SFII and the number of TRAP stained multinucleated osteoclasts was analyzed.

As a result, osteoclast formation was significantly inhibited when SFII (2 μM) was treated with RANKL (50 ng / ml) (E0), and osteoclasts when exposed to SFII after RANKL 1 day treatment (E1). Formation was significantly inhibited. Similarly, when exposed after 2 days (E2), there was a significant difference in the number of osteoclasts of 10 or more nuclei but little effect on the number of osteoclasts of 3 or more nuclei (FIG. 17A). ).

Expression of NFATc1 and its target genes CK, MMP9, TRAP, CR and DC-STAMP under the same experimental conditions were also measured by real-time PCR (see Table 2 primer sequence). When SFII was treated with RANKL When (E0) and SFII were treated after RANKL 1 day treatment (E1), both expression was significantly inhibited, but only DC-STAMP gene was significantly affected by SFII (E2) exposed 2 days after RANKL treatment. Was suppressed (B of FIG. 17).

Also, in order to see the effect of SFII on the formation of acting ring and bone resorption under similar experimental conditions, BMM cells were treated with RANKL for 3 days to differentiate into osteoclasts, and then treated with SFII and SFII. The cells treated with RANKL were fixed as follows and stained with actining and nuclei or analyzed for bone resorption on dentine discs. The cells were fixed in PBS added with 3.7% formaldehyde and treated with 0.1% Triton X-100, followed by 20 minutes treatment with Alexa Fluor 488-phalloidin (Invitrogen), followed by DAPI (4 ', 6-diamidino-2-phenylindole). , Roche) and observed through a fluorescence microscope. In addition, the degree of bone resorption was obtained by removing the cells cultured on dentine disc (Immunodiagnostic Systems Ltd) using cotton tip, and staining the resorption pit with hematoxylin. (Media Cybernetics).

 As a result, when treated with RANKL, SFII completely inhibited the formation of actin ring and bone resorption by osteoclasts in dentine disc, and when treated with SFNK after treatment with RANKL for 3 days, It was confirmed that the size of the actining was reduced and bone absorption was also significantly inhibited (A, B and C in FIG. 18). These results indicate that SFII may inhibit osteoclast fusion and bone resorption.

< Experimental Example  17> SF Inhibition of Bone Damage by Inflammation of Ⅱ

In order to confirm the inhibitory effect of bone damage caused by the inflammation of SFII in animal models, the mouse skull was injected twice a day with an inflammatory inducer, LPS (Lipopolysaccharide, 12.5 mg / kg body weight). At this time, vehicle (10% DMAC + 10% Tween 80 + 80% distilled water) or SFII (2 mg / kg) was administered together, and after 5 days of the first injection, the skull was fixed with 4% paraformaldehyde, and then PBS washed to TRAP stain Then observed. In addition, decalcification was performed with 0.5 M ethylenediaminetetraacetic acid for 7 days, paraffin blocks were made and then cut and stained with TRAP and Hematoxilin.

As a result, there were many TRAP stained parts in the skull treated only with LPS, but the TRAP stained part was significantly reduced in the skull treated with SFII (FIG. 19A). In addition, as a result of analysis by TRAP staining of the divided skull after descaling, it was confirmed that the number of bone cavity and osteoclasts increased by LPS treatment was significantly reduced by SFII (Figs. 19B and C).

III. Dehydrocostus  Identify the efficacy of lactones

< Experimental Example  18> Dehydrocostus  Inhibiting osteoclast differentiation of lactones

Dehydrocostus lactone (hereinafter referred to as DHCL) contained in the present invention as an active ingredient was used in an experiment using CFN98720 / 447-43-0 manufactured by ChemFaces.

First, the inventors conducted experiments on BMM (Bone Marrow-Derived Macrophages) cells in order to confirm the inhibitory effect of DHCL osteoclast differentiation.

BMM cells are precursor cells capable of differentiating into osteoclasts. In this experiment, cells extracted from femur and tibia bone marrow of 8-week-old C57BL / 6 male mice were used.

Bone Marrow-Derived Macrophages (BMM) cells were treated with 25 ng / ml M-CSF (macrophage colony stimulatory factor) and 50 ng / ml RANKL in the presence of various concentrations (0, 0.5, 1, 1.5 μM) of DHCL. After treatment for 1, 5 days to differentiate into osteoclasts, fixed with 4% paraformaldehyde after PBS washing, and then TRAP stained using leukocyte acid phosphatase cytochemistry kit (SigmaAldrich), followed by TRAP staining of osteoclasts with three or more multinucleated cells. The numbers were analyzed under a microscope.

As a result, when RANKL was treated for 4 days to differentiate into osteoclasts, osteoclast differentiation was significantly inhibited from 0.5 μM of DHCL (A and B in FIG. 20), and 0.5 μM of DHCL was also treated when treated for 5 days. Osteoclast differentiation was significantly inhibited (D and E in FIG. 20).

In addition, cytotoxicity was analyzed by EASY Cytox (WST-1) assay kit to exclude that the osteoclast differentiation inhibitory effect of DHCL is due to cytotoxicity. 10 μl of 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium monosodium salt (WST-1) reagent (Roche Applied Science) After incubation at 37 ° C. for 2-4 hours, absorbance at 450 nm was measured.

As a result, when treated with various concentrations of DHCL (0, 0.5, 1, 1.5 μM) for 4 days, 5 days, it was confirmed that no cytotoxicity by DHCL up to 1.5 μM concentration (Fig. 20 C and F) . This shows that the inhibition of osteoclast differentiation of DHCL is not due to apoptosis.

< Experimental Example  19> On DHCL  by NFATc1  Activation Suppression

To confirm the inhibitory effect of DHCL on osteoclast differentiation, BMM cells were treated with 50 ng / ml RANKL for 2 days in the presence of various concentrations (0, 0.5, 1, 1.5 μM) of DHCL to differentiate into osteoclasts, and osteoclasts. In order to confirm the expression of NFATc1 protein, the most important transcription factor in cell 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 nonspecific binding sites on the membrane were blocked using 5% skim milk dissolved in TBST (0.05% Tween-20 in Tris-buffered saline, pH 7.4). Primary antibodies (mouse monoclonal antibodies, Santa Cruz Biotechnology Inc) were treated at 4 ° C. for 14 hours and secondary antibodies were treated at room temperature for 1 hour and analyzed by West save Up (Ab Frontier).

As a result, it was confirmed that NFATc1 protein was significantly suppressed from 1 μM DHCL concentration (FIG. 21A), and the inhibitory effect was continued up to 4 days (FIG. 21B).

In addition, mRNA expression changes of DC-STAMP genes associated with CK, MMP9 and TRAP genes, which are target genes of NFATc1, and CR and osteoclasts, which are calcitonin receptor genes, were confirmed by real time-PCR.

For real time-PCR, total RNA was extracted using Trizol reagent (Invitrogen) and quantified at absorbance at 260 nm. Total RNA was adjusted to 15 ml total containing 0.5 mg oligo (dT) primers. After 5 minutes incubation at ℃, it was cooled directly on ice. The first strand of cDNA was synthesized at 25 μl for 1 hour at 42 ° C. and 10 minutes at 70 ° C. at 25 μl of final volume containing M-MLV reverse transcriptase (Promega) 200 units, ribonuclease inhibitor 24 units and 0.25 mM each of dNTP. ABI 7300 real at the final 20 μl volume containing 0.8 μl of the diluted cDNA template (1: 2.5) containing 10 μl of 2X SYBR Green PCR Master Mix (M Biotech) and 1 μl of 10 μM of each gene-specific primer (Genotech). Amplification was carried out using a time PCR system (Applied Biosystems), and the amplification was performed by performing 40 cycles for 2 minutes at 50 ° C, 2 minutes at 95 ° C, then 15 seconds at 95 ° C, and 1 minute at 60 ° C. The sequence of Primer used in the experiment is shown in Table 3 below.

real-time PCR  the sequence of the primer and amplicon  size

Gene		Primer (sense & antisense)	SEQ ID NO:	Amplicon length (bp)
36	NFATc1	5'-CTTCAGCTGGAGGACACC-3 '	71	79
		5'-CCAATGAACAGCTGTAGCG-3 '	72
37	CK	5'-ACCACTGCCTTCCAATACG-3 '	73	99
		5’-CGTGGCGTTATACATACAAC-3 ’	74
38	MMP9	5’-AGACGACATAGACGGCATC-3 ’	75	97
		5’-TGCTGTCGGCTGTGGTTC-3 ’	76
39	TRAP	5'-CCAGCGACAAGAGGTTCC-3 '	77	113
		5'-AGAGACGTTGCCAAGGTGAT-3 '	78
40	SOD2	5'-ATTAACGCGCAGATCATGCA-3 '	79	161
		5'-TGTCCCCCACCATTGAACTT-3 '	80
41	Catalase	5'-CTCGTTCAGGATGTGGTTTTC-3 '	81	145
		5'-CTTTCCCTTGGAGTATCTGGTG-3 '	82
42	c-Fos	5'-GAGAAACGGAGAATCCGAAG-3 '	83	97
		5'-GAGAAACGGAGAATCCGAAG-3 '	84
43	Nrf2	5'-TCTCCTCGCTGGAAAAAGAA-3 '	85	498
		5'-AATGTGCTGGCTGTGCTTTA-3 '	86
44	NQO1	5'-TTCTCTGGCCGATTCAGAG-3 '	87	180
		5'-GGCTGCTTGGAGCAAAATAG-3 '	88
45	Srx	5'-AGCCTGGTGGACACGATC-3 '	89	97
		5'-AGGAATAGTAGTAGTCGCCA-3 '	90
46	β-actin	5'-ACCCTAAGGCCAACCGTG-3 '	91	81
		5'-GCCTGGATGGCTACGTAC-3 '	92
47	CR	5'-TGATGACTCTCAGGACAATG-3 '	93	87
		5'-ACTGGATCAATCTGTAGGAG-3 '	94
48	DC-STAMP	5'-TTATGTGTTTCCACGAAGCCCTA-3 '	95	141
		5'-ACAGAAGAGAGCAGGGCAACG-3 '	96
CK, cathepsin K; MMP9, matrix metalloprotease 9; SOD2, superoxide dismutase 2; COX2, cyclooxygenase 2

As a result, when treated with 50 ng / ml RANKL for 2 days in the presence of 1.5 μM DHCL, mRNA of CR and DC-STAMP genes were significantly suppressed in addition to NFATc1 and its target genes CK, MMP9 and TRAP. (FIG. 21C).

< Experimental Example  20> On DHCL  By c- FOS  control

To determine if DHCL regulates NF-κB activity and c-Fos expression, known as upstream regulators of NFATc1, immunoblot using IκBα and p-IκBα (rabbit polyclonal antibodies, Cell Signaling Technology) was used to determine the degree of phosphorylation of IκBα. Expression of SOD2 and c-FOS proteins was also confirmed by immunoblot using these antibodies (SOD2 / rabbit polyclonal antibody, Upstate; c-FOS / rabbit polyclonal antibody, Santa Cruz Biotechnology, Inc), and SOD2 and c-Fos. Expression of mRNA was analyzed by real-time PCR by the method described in Experimental Example 19 (see Table 3 primer sequence).

As a result, DHCL (1.5 μM) significantly inhibited the phosphorylation of IκBα in the presence of RANKL (50 ng / ml) (FIG. 22A) and significantly inhibited protein and mRNA expression of SOD2, an NF-κB target gene. (FIGS. 22B and D). In addition, it was confirmed that DHCL also significantly inhibits the expression of c-Fos protein and mRNA (C and D of Figure 22).

< Experimental Example  21> On DHCL  by MAPKs  And AKT  Active inhibition

To see the effect of DHCL on MAPKs and AKT activated by RANKL, Rabbit polyclonal against their antibodies (phospho-JNK, phospho-ERK, phospho-p38, phospho-Src, phospho-AKT, phospho-Foxo1, Src) We compared the phosphorylation levels of MAPKs and AKT-related proteins by immunoblot using rabbit monoclonal antibodies against AKT, Foxo1, Cell Signaling Technology; Rabbit polyclonal antibodies against JNK1, p38, ERK2 mouse monoclonal antibody, Santa Cruz Biotechnology, Inc. saw.

As a result, it was confirmed that phosphorylation of JNK and ERK was slightly inhibited by DHCL and that phosphorylation of Src-AKT was significantly suppressed (A and B in FIG. 23). In addition, AKT phosphorylates FOXO1, which induces degradation and inhibits migration to the nucleus, and further confirms the effect of DHCL on phosphorylation of FOXO1. . DHCL inhibited AKT activity and inhibited phosphorylation of FOXO1. As a result, it was confirmed that the degradation of FOXO1 was also inhibited (FIG. 23C). As a result, the transcriptional activity of FOXO1 was increased and the expression of catalase, a target gene, was increased at both protein and mRNA levels by immunoblot and real-time PCR (FIG. 23 C and D).

< Experimental Example  22> On DHCL  by Nrf2  Activation and free radical regulation

Redox-sensitive transcription factor Nrf2 is known to be involved in osteoclast differentiation by regulating the expression of GSH synthase and antioxidant enzymes in vivo, and to investigate the inhibitory effect of osteoclast differentiation by regulating Nrf2 activity of DHCL. , Nrf2, NQO1 and Srx gene expression was confirmed by real-time PCR (see Table 3 primer sequence), and target proteins of Nrf2 including Nrf2 and antioxidant enzymes NQO1, Srx, Prx, PrxV, PrxVI and Trx1 (rabbit polyclonal antibodies, Young In Frontier) protein expression 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 DHCL (1.5 μM) treatment in the presence of RANKL (50 ng / ml) (FIG. 24A). In addition, Nrf2 and antioxidant enzymes, including its target proteins NQO1, Srx, Prx I, PrxV, PrxVI and Trx1 was also confirmed that the expression is increased (Fig. 24B).

In addition, it is well known that free radicals are produced and involved in the differentiation process of osteoclasts by RANKL. If DHCL activates Nrf2 and the expression of Nrf2 target proteins including antioxidant enzymes is increased, these antioxidant enzymes may be involved in RANKL. In order to confirm this possibility, the active oxygen generated by the reaction may be removed, and RANKL was treated for 2 days in the presence of DHCL and the reactive oxygen species (ROS) were measured.

ROS was measured by oxidant sensitive fluorescence detection using CM-H2DCFDA. Since CM-H2DCFDA reacts with ROS and fluoresces, the cells are treated with RANKL, washed with HBSS, and then treated with 5 μM of CM-H2DCFDA for 20 minutes in the dark, using a FACS Calibur flow cytometer (BD Biosciences). Analyzed.

As a result, it was confirmed that the free radicals in the cell significantly decreased in the presence of DHCL (FIG. 24C, left).

In addition, DHCL (1.5 μM) was pretreated for 30 minutes and RANKL (C in FIG. 24) or H ₂ O ₂ (C in FIG. 24) to determine the possibility of DHCL being able to directly remove active oxygen as an antioxidant. After treating for 15 minutes, the right oxygen was measured using CM-H2DCFDA as described above.

As a result, it was confirmed that free radicals in cells are reduced by DHCL (C, middle and right in Fig. 24). Since DHCL was pretreated only for a short time before the expression of antioxidant enzymes by Nrf2 activation, this result means that free radicals may be directly removed by DHCL.

In addition, since caveolin-1 regulates the expression of Nrf2 and is known as a promoter of osteoclast differentiation, the effects of DHCL on the expression of caveolin-1 have been tested. As a result, DHCL is caveolin-1 (rabbit monoclonal) induced by RANKL. It was confirmed that the expression of antibody, Trnasduction Laboratories) is suppressed (FIG. 24D). This suggests that DHCL inhibits the expression of caveolin-1, thereby reducing the binding of Nrf2 to promote Nrf2 activity, resulting in lowering free radicals in cells.

< Experimental Example  23> On DHCL  Inhibition of bone resorption by osteoclasts

In order to investigate the differentiation inhibitory effect of DHCL osteoclasts, different time points (E0, RANKL treatment; E1, RANKL 1 day treatment; E2, RANKL 2 days) during RANKL treatment to differentiate BMM cells into osteoclasts After treatment; after 3 days of treatment with E3, RANKL), DHCL was exposed and the number of TRAP stained multinucleated osteoclasts was analyzed.

As a result, when DHCL (1.5 μM) was treated with RANKL (50 ng / ml) (E0) and RANKL one-day treatment (E1) and two-day treatment (E2), which can be called early differentiation of osteoclasts, The formation of osteoclasts was significantly inhibited, but in E3 treated with mature osteoclasts, E3 had little effect on the number of osteoclasts of three or more nuclei (FIG. 25A). However, the number of osteoclasts above 10 nuclei was slightly reduced.

Under the same experimental conditions, mRNA expression of NFATc1, its target genes MMP9, TRAP and DC-STAMP genes, CK and CR was also measured by real-time PCR (see Table 3 primer sequence), indicating that NFATc1 and its target genes Expression was also inhibited at the beginning of the differentiation of osteoclasts but hardly inhibited in mature osteoclasts (FIG. 25B).

In addition, in order to see the effect of DHCL on the formation of acting ring and bone resorption under similar experimental conditions, BMM cells were treated with RANKL for 3 days to differentiate into osteoclasts, and then treated with DHCL and DHCL. The cells treated with RANKL were fixed as follows and stained with actining and nuclei or analyzed for bone resorption on dentine discs. The cells were fixed in PBS with 3.7% formaldehyde and treated with 0.1% Triton X-100, followed by treatment with Alexa Fluor 488-phalloidin (Invitrogen) for 30 minutes, followed by DAPI (4 ', 6-diamidino-2-phenylindole). , Roche) and observed through a fluorescence microscope. In addition, the degree of bone resorption was obtained by removing the cells cultured on dentine disc (Immunodiagnostic Systems Ltd) using cotton tip, and staining the resorption pit with hematoxylin. (Media Cybernetics).

 As a result, when treated with RANKL, DHCL completely inhibited the formation of actin ring and bone resorption by osteoclasts in dentine disc, and when treated with RANKL for 3 days to differentiate into osteoclasts, It was confirmed that the size of the actining was reduced and bone absorption was also significantly inhibited (A, B and C in Fig. 26). These results indicate that DHCL may inhibit osteoclast fusion and bone resorption.

< Experimental Example  24> Of DHCL  Suppress bone damage caused by inflammation

In order to confirm the inhibitory effect of bone damage caused by inflammation of DHCL in an animal model, LPS (Lipopolysaccharide, 12.5 mg / kg body weight), an inflammatory inducer, was injected into the mouse skull twice daily at intervals. At this time, vehicle (10% DMAC + 10% Tween 80 + 80% distilled water) or DHCL (2 mg / kg) was administered together, and after 5 days of the first injection, the skull was fixed with 4% paraformaldehyde, followed by PBS washing to TRAP staining. Then observed. In addition, decalcification was performed with 0.5 M ethylenediaminetetraacetic acid for 7 days, paraffin blocks were made and then cut and stained with TRAP and Hematoxylin.

As a result, there were many TRAP stained parts in the skull treated only with LPS, but the TRAP stained part was significantly reduced in the skull treated with the DHCL (FIG. 27A). In addition, as a result of quantitative analysis, it was confirmed that the number of bone cavity and osteoclasts increased by LPS treatment was significantly reduced by DHCL (FIG. 27B and C).

Ⅳ. Neoconinic  Efficacy Check

< Experimental Example  25> Neoconinic  Osteoclast differentiation inhibition

Synconin (hereinafter referred to as CN) contained in the present invention as an active ingredient was used in an experiment using 27370 / 118-10-5 product of Sigma-Aldrich.

First, in order to confirm the inhibitory effect on the osteoclast differentiation of Cinconin, 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 extracted from femur and tibia bone marrow of 8-week-old C57BL / 6 male mice were used.

 25 ng / ml M-CSF (macrophage colony stimulating factor) and 50 ng / ml RANKL (Bone Marrow-Derived Macrophages) cells in the presence of various concentrations (0, 10, 20, 30 μM) PeproTech Inc) was treated for 3 days to differentiate into osteoclasts, fixed with 4% paraformaldehyde after PBS washing, and then TRAP stained using leukocyte acid phosphatase cytochemistry kit (SigmaAldrich), followed by TRAP staining. The number of osteoclasts was analyzed under a microscope.

As a result, osteoclast differentiation was effectively suppressed from 20 μM of cinconin (FIGS. 28A and 28B).

In addition, the cytotoxicity was analyzed by EASY Cytox (WST-1) assay kit to exclude the fact that the inhibition of osteoclast differentiation of the cytokine is caused by cytotoxicity. BMM cells were prepared using 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium mono-sodium salt (WST-1) reagent (Roche Applied Science), respectively. μl was added 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 caused by synconin was shown up to a concentration of 30 μM (FIG. 28C). In addition, osteoclast differentiation was significantly inhibited even when BMM cells were differentiated by treatment with RANKL for 3 to 5 days in the presence of 20 μM cinconin (FIG. 28D and E in FIG. 28), and cytotoxicity was shown up to 5 days. It was confirmed that no (FIG. 28F). This shows that the effect of inhibiting osteoclast differentiation of synconin is not due to apoptosis.

< Experimental Example  26> In shinkonin  by NFATc1  Activation Suppression

In order to confirm the inhibitory effect on the osteoclast differentiation of Cinconin, BMM cells were treated with 50 ng / ml of RANKL for 2 days in the presence of 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 nonspecific binding sites on the membrane were blocked using 5% skim milk dissolved in TBST (0.05% Tween-20 in Tris-buffered saline, pH 7.4). Primary antibodies (mouse monoclonal antibodies, Santa Cruz Biotechnology Inc) were treated at 4 ° C. for 14 hours and secondary antibodies were treated at room temperature for 1 hour and analyzed by West save Up (Ab Frontier).

As a result, the NFATc1 protein was significantly inhibited from 20 μM succinonin concentration (FIG. 29A), and it was confirmed that the inhibitory effect was continued up to 5 days (FIG. 29B).

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

For real time-PCR, total RNA was extracted using Trizol reagent (Invitrogen) and quantified at absorbance at 260 nm. Total RNA was adjusted to 15 μl, containing 0.5 mg of oligo (dT) primers. After incubation for 5 minutes at C, it was cooled directly on ice. The first strand of cDNA was synthesized at 25 μl for 1 hour at 42 ° C. and 10 minutes at 70 ° C. at 25 μl of final volume containing M-MLV reverse transcriptase (Promega) 200 units, ribonuclease inhibitor 24 units and 0.25 mM each of dNTP. ABI 7300 real at the final 20 μl volume containing 0.8 μl of the diluted cDNA template (1: 2.5) containing 10 μl of 2X SYBR Green PCR Master Mix (M Biotech) and 1 μl of 10 μM of each gene-specific primer (Genotech). Amplification was carried out using a time PCR system (Applied Biosystems), and the amplification was performed by performing 40 cycles for 2 minutes at 50 ° C, 2 minutes at 95 ° C, then 15 seconds at 95 ° C, and 1 minute at 60 ° C. The sequence of Primer used in the experiment is shown in Table 4.

real-time PCR  the sequence of the primer and amplicon  size

Gene		Primer (sense & antisense)	SEQ ID NO:	Amplicon length (bp)
49	NFATc1	5'-CTTCAGCTGGAGGACACC-3 '	97	79
		5'-CCAATGAACAGCTGTAGCG-3 '	98
50	CK	5'-ACCACTGCCTTCCAATACG-3 '	99	99
		5’-CGTGGCGTTATACATACAAC-3 ’	100
51	MMP9	5’-AGACGACATAGACGGCATC-3 ’	101	97
		5’-TGCTGTCGGCTGTGGTTC-3 ’	102
52	TRAP	5'-CCAGCGACAAGAGGTTCC-3 '	103	113
		5'-AGAGACGTTGCCAAGGTGAT-3 '	104
53	SOD2	5'-ATTAACGCGCAGATCATGCA-3 '	105	161
		5'-TGTCCCCCACCATTGAACTT-3 '	106
54	COX2	5'-GATCATAAGCGAGGACCTG-3 '	107	85
		5'-GTCTGTCCAGAGTTTCACC-3 '	108
55	c-Fos	5'-GAGAAACGGAGAATCCGAAG-3 '	109	97
		5'-GAGAAACGGAGAATCCGAAG-3 '	110
56	PGC-1β	5'-CTCCAGGCAGGTTCAACCC-3 '	111	83
		5'-GGGCCAGAAGTTCCCTTAGG-3 '	112
57	ND4	5'-CATCACTCCTATTCTGCCTAGCAA-3 '	113	74
		5'-TCCTCGGGCCATGATTATAGTAC-3 '	114
58	COX1	5'-TTTTCAGGCTTCACCCTAGATGA-3 '	115	81
		5'-GAAGAATGTTATGTTTACTCCTACGAATATG-3 '	116
59	COX3	5'-CGGAAGTATTTTTCTTTGCAGGAT-3 '	117	82
		5'-CAGCAGCCTCCTAGATCATGTG-3 '	118
60	Cytb	5'-GCCACCTTGACCCGATTCT-3 '	119	64
		5'-TTGCTAGGGCCGCGATAAT-3 '	120
61	β-actin	5'-ACCCTAAGGCCAACCGTG-3 '	121	81
		5'-GCCTGGATGGCTACGTAC-3 '	122
62	Catalase	5'-CTCGTTCAGGATGTGGTTTTC-3 '	123	145
		5'-CTTTCCCTTGGAGTATCTGGTG-3 '	124
CK, cathepsin K; MMP9, matrix metalloprotease 9; SOD2, superoxide dismutase 2; COX2, cyclooxygenase 2

As a result, when treated with 50 ng / ml RANKL for 2 days in the presence of 20 μM myconin, it was confirmed that the mRNA expression of NFATc1 and its target genes CK, MMP9 and TRAP genes are significantly suppressed (Fig. 29 C). ).

< Experimental Example  27> In shinkonin  by NF - κB  And c- FOS  control

Phosphorylation of IκBα by immunoblot using antibodies to IκBα and p-IκBα (Rabbit polyclonal antibodies, Cell Signaling Technology) to determine whether Cinconin regulates NF-κB activity and c-Fos expression, known as upstream regulators of NFATc1 SOD2 and c-FOS protein expression was also confirmed by immunoblot using antibodies (SOD2 / rabbit polyclonal antibody, Upstate; c-FOS / Rabbit polyclonal antibody, Santa Cruz Biotechnology Inc), and SOD2 and c-Fos mRNA expression was confirmed. It was analyzed by real-time PCR by the method described in Experimental Example 26 (see Table 4 primer sequence).

As a result, Cinconin (20 μM) significantly inhibited the phosphorylation of IκBα in the presence of RANKL (50 ng / ml) (FIG. 30A), and the protein and mRNA expression of SOD2 and COX2, NF-κB target genes, were also significant. Inhibition (B and D of FIG. 30). In addition, it was confirmed that cinnacon also significantly inhibited c-Fos protein and mRNA expression (C and D of FIG. 30).

< Experimental Example  28> In shinkonin  by ERK  And Src - AKT  Active inhibition

To see the effect of Cinconin on MAPKs and AKT activated by RANKL, Rabbits on their antibodies (phospho-JNK, phospho-ERK, phospho-p38, phospho-Src, phospho-AKT, phospho-Foxo1, Src) Polyclonal antibodies, AKT, rabbit monoclonal antibodies against Foxo1, Cell Signaling Technology; JNK1, rabbit polyclonal antibodies against p38, ERK2 mouse monoclonal antibody, Santa Cruz Biotechnology, Inc. Compared.

As a result, it was confirmed that phosphorylation of both ERK and Src-AKT is suppressed by syntholin in MAPKs (A and B of FIG. 31). In addition, AKT phosphorylates FOXO1, which induces degradation and inhibits migration to the nucleus. Thus, as a result of confirming the effect of synconin on the phosphorylation of FOXO1, it was confirmed that the phosphorylation of FOXO1 was inhibited (FIG. 31B). ). Cinconin inhibited the phosphorylation of FOXO1 by inhibiting AKT activity, thereby confirming that the degradation of FOXO1 was also inhibited (FIG. 31C). As a result, it was confirmed by immunoblot and real-time PCR that the expression of catalase, which is a target gene, increased in FOXO1 transcriptional activity, at both protein and mRNA levels (FIG. 31C and D).

< Experimental Example  29> In shinkonin  by PGC - 1β  Active inhibition

PGC-1β is a transcription factor well known to be activated by RANKL and involved in osteoclast differentiation. In order to confirm the effect of PGC-1β regulation of cinconin, mRNA expression of PGC-1β and its target genes ND4, Cyt b, COX1 and COX3 was confirmed by real-time PCR (see Table 4 primer sequence).

As a result, cinconin (20 μM) significantly inhibited PGC-1β, ND4, Cyt b, COX1 and COX3 mRNA expression by RANKL (50 ng / ml) (FIG. 32A). In addition, Cinconin inhibited the expression of PGC-1β (rabbit polyclonal antibody, Abcam) protein, but CREB (phospho-CREB rabbit monoclonal antibody, Cell Signaling Technology; CREB mouse monoclonal antibody, Santa Cruz, a higher transcription factor of PGC-1β) Biotechnology, Inc) did not affect the phosphorylation (B and C of Figure 32).

< Experimental Example  30> In shinkonin  Inhibition of bone resorption by osteoclasts

To investigate the differentiation-inhibiting time of the osteoclasts of Cinconin, during differentiation of BMM cells into osteoclasts by treatment of RANKL, several time points (E0, RANKL treatment; E1, RANKL after 1 day treatment; E2, RANKL 2 After one treatment), the number of multinucleated osteoclasts stained with cinconin and TRAP stained.

As a result, when the treatment with cinnaconin (20 μM) with RANKL (50 ng / ml) (E0) almost suppressed the formation of osteoclasts, when treated with synonin after RANKL 1 day treatment (E1) Significant inhibition was observed, but little effect on osteoclast formation when treated with synonin after 2 days (E2) (FIG. 33A).

Expression of NFATc1 and its target genes CK, MMP9, TRAP and PGC-1β and its target genes ND4, COX1, COX3, and Cyt b genes under the same experimental conditions were also measured by real-time PCR (Table 4). The expression was significantly inhibited when Cynconin was treated with RANKL (E0), but was hardly inhibited by Cynonin exposed after RANKL 2 days treatment (E2) (Fig. 33). B and c).

In addition, in order to see the effect of synonin on the formation of actin ring (acting ring) and bone resorption under similar experimental conditions, BMM cells were treated with RANKL for 3 days to differentiate into osteoclasts, Cells treated with Cinconin with RANKL were fixed as follows and stained for actining and nuclei or analyzed for bone resorption on dentine discs. The cells were fixed in PBS with 3.7% formaldehyde and treated with 0.1% Triton X-100, followed by Alexa Fluor 488-phalloidin (Invitrogen) for 20 minutes, followed by DAPI (4 ', 6-diamidino-2-phenylindole). , Roche) and observed through a fluorescence microscope. In addition, the degree of bone resorption was obtained by removing the cells cultured on dentine disc (Immunodiagnostic Systems Ltd) using cotton tip, and staining the resorption pit with hematoxylin. (Media Cybernetics).

 As a result, when treated with RANKL, synonin inhibited the formation of acting ring and bone uptake, but did not inhibit actining formation and bone uptake in cells differentiated with RANKL for 3 days (Fig. 33). D and F).

These results show that cinconin may inhibit osteoclast formation and bone resorption.

< Experimental Example  31> Neoconinic  Suppress bone damage caused by inflammation

In order to confirm the inhibitory effect of bone damage caused by the inflammation of cinnaconin in an animal model, the mouse skull was infused with LPS (Lipopolysaccharide, 12.5 mg / kg body weight), which is an inflammation-inducing substance, twice daily at intervals. At this time, vehicle (20% polyethylene glycol + 20% ethanol + 60% distilled water) or cinconin (10 mg / kg) was administered together, and 5 days after the first injection, the skull was fixed with 4% paraformaldehyde and PBS washed. Decalcification was performed with 0.5 M ethylenediaminetetraacetic acid for 7 days, paraffin blocks were made, cut and stained with TRAP and hematoxilin for observation.

As a result, it was confirmed that the number of bone cavities and osteoclasts increased by LPS induction was significantly reduced by Cinconin (FIGS. 34A and 34).

Claims (43)

1. A pharmaceutical composition for preventing or treating bone diseases, comprising Euphorbia Factor L1 (Euphorbia Factor L1) or a pharmaceutically acceptable salt thereof as an active ingredient, represented by Formula 1 below:

   [Formula 1]

   
2. The pharmaceutical composition for preventing or treating bone disease according to claim 1, wherein the Euphorbia factor L1 is in a concentration of 0.1 to 20 μM.
3. According to claim 1, wherein the composition is a pharmaceutical composition for the prevention or treatment of bone diseases, characterized in that the expression or activity of NFATc1, NF-κB, c-FOS and PGC-1β reduces.
4. According to claim 1, wherein the composition is a pharmaceutical composition for preventing or treating bone diseases, characterized in that for increasing the expression or activity of Nrf2.
5. According to claim 1, wherein the composition is a pharmaceutical composition for preventing or treating bone diseases, characterized in that the production of free radicals in the cell.
6. According to claim 1, wherein the composition is a pharmaceutical composition for the prevention or treatment of bone diseases, characterized in that to promote the death of differentiated osteoclasts.
7. According to claim 1, wherein the composition is a pharmaceutical composition for preventing or treating bone diseases, characterized in that to inhibit bone damage caused by inflammation.
8. The method of claim 1, wherein the bone disease is a growth stage of development, fracture, osteoporosis (osteoporosis) due to excessive bone resorption of osteoclasts (rheumatoid arthritis), periodontal disease (periodontal disease), Paget disease (Paget disease) ) And metastatic bone cancers (metastatic bone cancers), any one or more selected from the group consisting of a pharmaceutical composition for the prevention or treatment of bone diseases.
9. Euphorbia Factor L1 (Euphorbia Factor L1) or a pharmaceutically acceptable salt thereof as an active ingredient, which is represented by the formula (1):

   [Formula 1]

   
10. 10. The method of claim 9, wherein the bone disease is a growth stage development, fracture, osteoporosis (osteoporosis) due to excessive bone resorption of osteoclasts (rheumatoid arthritis), periodontal disease (periodontal disease), Paget disease (Paget disease) ) And metastatic bone cancers (metastatic bone cancers) health functional food for improvement of bone disease, characterized in that any one or more selected from the group consisting of.
11. A method of preventing or treating bone disease, comprising administering Euphorbia Factor L1 or a pharmaceutically acceptable salt thereof to a mammal, which is represented by Formula 1 below:

    [Formula 1]

    
12. Use of Euphorbia Factor L1, or a pharmaceutically acceptable salt thereof, represented by Formula 1 for use in the manufacture of a medicament for the prevention or treatment of bone disease:

    [Formula 1]

    
13. A pharmaceutical composition for preventing or treating bone diseases, comprising as an active ingredient a scullcapflavone derivative represented by Formula 2 or a pharmaceutically acceptable salt thereof:

    [Formula 2]

    

    Wherein R ₁ and R ₂ are H or OCH ₃ .
14. The pharmaceutical composition for preventing or treating bone disease according to claim 13, wherein the skull capflavone derivative is skull capflavone I or skull capflavone II.
15. The pharmaceutical composition for preventing or treating bone disease according to claim 13, wherein the skullcapflavone derivative is at a concentration of 0.1 to 10 μM.
16. The pharmaceutical composition for preventing or treating bone diseases of claim 13, wherein the composition reduces the expression or activity of NFATc1, c-FOS, MAPK _S , Src, AKT, CREB, PGC-1β and caveolin-1. Composition.
17. The pharmaceutical composition for preventing or treating bone disease according to claim 13, wherein the composition increases the expression or activity of Nrf2, FOXO1 and catalase.
18. The method of claim 13, wherein the composition is a pharmaceutical composition for preventing or treating bone diseases, characterized in that the production of free radicals in the cell.
19. According to claim 13, wherein the composition is a pharmaceutical composition for the prevention or treatment of bone diseases, characterized in that to inhibit bone damage caused by inflammation.
20. The method of claim 13, wherein the bone disease is a growth stage of development, fracture, osteoporosis (osteoporosis) due to excessive bone resorption of osteoclasts (rheumatoid arthritis), periodontal disease (periodontal disease), Paget disease (Paget disease) ) And metastatic bone cancers (metastatic bone cancers) is a pharmaceutical composition for the prevention or treatment of bone diseases, characterized in that at least one selected from the group consisting of.
21. A health functional food for improving bone disease, comprising as an active ingredient a scull capflavone derivative or a pharmaceutically acceptable salt thereof represented by Formula 2 below:

    [Formula 2]

    

    Wherein R ₁ and R ₂ are H or OCH ₃ .
22. 22. The health functional food for improving bone disease according to claim 21, wherein the skull capflavone derivative is Skull cap Flavon I or Skull cap Flavon II.
23. 22. The method of claim 21, wherein the bone disease is a growth stage of development, fracture, osteoporosis (osteoporosis) due to excessive bone resorption of osteoclasts (rheumatoid arthritis), periodontal disease (periodontal disease), Paget disease (Paget disease) ) And metastatic bone cancers (metastatic bone cancers) health functional food for improving bone disease, characterized in that at least one selected from the group consisting of.
24. A method for preventing or treating bone disease, comprising administering to a mammal a skullcapflavone derivative represented by Formula 2 or a pharmaceutically acceptable salt thereof:

    [Formula 2]

    

    Wherein R ₁ and R ₂ are H or OCH ₃ .
25. Use of a skullcapflavone derivative represented by the following formula (2) or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for the prevention or treatment of bone disease:

    [Formula 2]

    

    Wherein R ₁ and R ₂ are H or OCH ₃ .
26. A pharmaceutical composition for preventing or treating bone diseases, characterized in that it contains dihydrocostus lactone represented by Formula 5 or a pharmaceutically acceptable salt thereof as an active ingredient:

    [Formula 5]

    
27. The pharmaceutical composition for preventing or treating bone disease of claim 26, wherein the dihydrocostus lactone is in a concentration of 0.1 to 10 μM.
28. The pharmaceutical composition of claim 26, wherein the composition reduces the expression or activity of NFATc1, c-FOS, JNK, ERK, Src, AKT, and caveolin-1.
29. The pharmaceutical composition of claim 26, wherein the composition inhibits bone damage caused by inflammation.
30. 27. The method of claim 26, wherein the bone disease is a growth stage of development, fracture, osteoporosis (osteoporosis) due to excessive bone resorption of osteoclasts (rheumatoid arthritis), periodontal disease (periodontal disease), Paget disease (Paget disease) ) And metastatic bone cancers (metastatic bone cancers), any one or more selected from the group consisting of a pharmaceutical composition for the prevention or treatment of bone diseases.
31. A dietary supplement for improving bone disease, characterized in that it contains dihydrocostus lactone represented by the following formula (5) or a pharmaceutically acceptable salt thereof as an active ingredient:

    [Formula 5]

    
32. 32. The method of claim 31, wherein the bone disease is a growth stage development, fracture, osteoporosis (osteoporosis) due to excessive bone resorption of osteoclasts (rheumatoid arthritis), periodontal disease (periodontal disease), Paget disease (Paget disease) ) And metastatic bone cancers (metastatic bone cancers) health functional food for improvement of bone disease, characterized in that any one or more selected from the group consisting of.
33. A method for preventing or treating bone diseases, comprising administering to a mammal a dihydrocostus lactone represented by Formula 5 or a pharmaceutically acceptable salt thereof:

    [Formula 5]

    
34. Use of dihydrocostus lactone or a pharmaceutically acceptable salt thereof represented by Formula 5 for use in the manufacture of a medicament for the prevention or treatment of bone disease:

    [Formula 5]

    
35. A pharmaceutical composition for the prevention or treatment of bone diseases, which contains as a active ingredient a cinnaconin or a pharmaceutically acceptable salt thereof represented by the following formula (6):

    [Formula 6]

    
36. 36. The pharmaceutical composition for preventing or treating bone disease according to claim 35, wherein the cinconin is in a concentration of 0.1 to 50 µM.
37. The pharmaceutical composition for preventing or treating bone disease of claim 35, wherein the composition reduces the expression or activity of NFATc1, NF-κB, ERK, Src, AKT, FOXO1, c-FOS, and PGC-1β. Composition.
38. 36. The pharmaceutical composition for preventing or treating bone disease according to claim 35, wherein the composition inhibits bone damage caused by inflammation.
39. 36. The method of claim 35, wherein the bone disease is a growth stage development, fracture, osteoporosis (osteoporosis) due to excessive bone resorption of osteoclasts (rheumatoid arthritis), periodontal disease (periodontal disease), Paget disease (Paget disease) ) And metastatic bone cancers (metastatic bone cancers), any one or more selected from the group consisting of a pharmaceutical composition for the prevention or treatment of bone diseases.
40. A health functional food for improving bone disease, which contains as a active ingredient a cinnaconin or a pharmaceutically acceptable salt thereof represented by the following formula (6):

    [Formula 6]

    
41. 41. The method of claim 40, wherein the bone disease is a growth stage of development, fractures, osteoporosis (osteoporosis) due to excessive bone resorption of osteoclasts (rheumatoid arthritis), periodontal disease (periodontal disease), Paget disease (Paget disease) ) And metastatic bone cancers (metastatic bone cancers) health functional food for improvement of bone disease, characterized in that any one or more selected from the group consisting of.
42. A method for preventing or treating bone disease, comprising administering to a mammal a neoconsin or a pharmaceutically acceptable salt thereof represented by Formula 6 below:

    [Formula 6]

    
43. Use of a Cinconin, or a pharmaceutically acceptable salt thereof, as described in Formula 6 for use in the manufacture of a medicament for the prevention or treatment of bone disease:

    [Formula 6]

    

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