KR20150055667A - Comoposition for Preventing or Treating Osteoporosis Comprising Extract Containing Scoparone Derived from Artemisia Capillaris - Google Patents

Abstract

The present invention relates to a composition which includes a wormwood (Artemisia capillaris) extract containing scoparone as an active ingredient and is used for the prevention and treatment of osteoporosis. More specifically, the present invention relates to the composition which includes the scoparone or the wormwood extract containing the scoparone as an active ingredient and a dietary supplement product used for the prevention and treatment of osteoporosis. According to the present invention, the scoparone or the wormwood extract containing the scoparone can increase the osteoblast activity and inhibit the osteoclast activity and bone resorption activity. Accordingly, the present invention can be used as a pharmaceutical composition, dietary supplement product, or beverage to improve the health of the bones and prevent bone loss as well as for the prevention and treatment of osteoporosis.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating osteoporosis, which comprises extracts containing scoparone derived from dead sparrows as an effective ingredient. [0002] The present invention relates to a composition for preventing or treating osteoporosis,

The present invention relates to a process for the production of artemisia The present invention relates to a composition for preventing or treating osteoporosis comprising an extract containing scoparone derived from capillaris as an active ingredient and more particularly to a composition for prevention or treatment of osteoporosis comprising scoparone or the above- Therapeutic compositions and health functional foods.

Bone tissue is a dynamic tissue that is constantly absorbed and formed to maintain bone mass and skeletal homeostasis. Two types of specialized cells are involved in bone remodeling. do. While osteoclasts absorb bone, osteoblasts play a role in synthesizing and filling bone matrix, so bone mass depends on the relative function of these cells. In normal adults, there is always a balance between the amount of bone resorption and the amount of bone formation. The bone remodeling takes place over a period of time and occurs locally at the narrow site to maintain the skeletal structure, which is regulated by the complex interactions between systemic hormones and local factors (Canalis, E. et al. meat al ., J. Clin . Invest ., 81: 277, 1988; Raisz, LG, CIBA Foundation Symposium , 136: 226, 1988).

Through the action of osteoblast and osteoclast, it constantly forms and absorbs for a lifetime and carries out the process of aggregate formation. These aggregating processes include hormones such as parathyroid hormone (PTH), calci-tonin, estrogen, and various growth factors secreted from bone tissue such as insulin-like growth factor I (IGF-I), tumor necrosis factor- ) To regulate the balance of osteoblast and osteoclast activity and maintain homeostasis. However, when genetic, physiological, and environmental factors are changed, osteoblast activity is weakened to decrease osteogenesis, osteoclast activity increases osteoporosis, or when two factors are simultaneously acting, . There are various theories and arguments as to whether this imbalance of bone metabolism is caused by a decrease in function of osteoblast cells or an increase in osteoclast activity or an increase in osteoclast activity. The factors of osteoporosis include age, Deficiency, lack of exercise, lack of calcium intake, genetic factors, drug use, and hormonal effects (Ryan PJ et al ., J Bone Miner Res . , 7: 1455,1992).

Osteoporosis, which is a major problem in aging society, is a disease caused by collapse of the balance between osteoporosis and osteogenesis. Osteoporosis is caused by excessive bone resorption rather than osteogenesis. Of the knee joint is thinned and thus the bone marrow becomes wider and the bone is weakened as the symptom progresses, so that even a small impact is likely to fracture. Bone tissue is a dynamic tissue that is formed by osteoblasts and is constantly repetitively absorbed and destroyed by osteoclasts.

Osteoporosis is a cause of 15% of the deaths of the elderly as a result of various fractures, especially femoral fractures or vertebral fractures, which are easily caused by the weakening of the bone rather than the symptom itself, . The bone mass is affected by various factors such as genetic factors, nutritional intake, hormonal changes, exercise and lifestyle differences, and the causes of osteoporosis include age, lack of exercise, low birth weight, smoking, low calcium diet, Ovariectomy and the like are known. Although there are individual differences, blacks have a lower bone resorption level than blacks, and bone mass is higher. Usually, bone mass is highest at 14-18 years old and decreases about 1% per year at old age. In particular, postmenopausal women are known to have the highest incidence of estrogen secretion (Jilka RL., Bone , 23:75, 1998).

However, even in men, bone mass tends to decrease with aging (Stein GS et al ., Physiol Rew . , 76: 593, 1996), and the fracture rate of elementary and junior high school students has more than doubled over the past 20 years. In terms of prevention or treatment of bone diseases due to bone metabolic imbalance, it is important to raise bone mass from a young age, and thus maintaining bone health is an important issue regardless of gender or age.

Currently, most of the drugs used for the prevention and treatment of osteoporosis are unable to completely recover the already existing bone loss because they inhibit the bone resorption. Thus, the ultimate goal of osteoporosis can not be completely prevented. However, there is a need for a scientific approach to osteoporosis prevention and treatment, and its effect on bone regeneration ability (Cho SH et al., Korean J Obstet Gynecol . , ≪ / RTI > 39: 1497, 1996; Boonene et al ., J Internal Medicine. , 242: 285,1997).

Estrogen, which is administered for the treatment of postmenopausal osteoporosis, binds to estrogen receptor α (ER α) or β (ER β) in osteoblasts in addition to sex hormone function in vivo, In addition to regulating the transcriptional activity of the locus factor gene, it is involved in bone formation by increasing the expression of collagen, the activity of alkaline phosphatase (ALP) involved in calcium and phosphorus metabolism, and the expression of IGF-I, . In addition, it stimulates osteoblast cells to promote calcification of bone, inhibits the secretion of RANK ligand (RANKL), which promotes osteoclastogenesis, and inhibits the production of new osteoclasts, while promoting apoptosis of osteoclast . However, long-term use has been reported to cause side effects such as nausea, weight gain, irregular uterine bleeding, endometrial cancer, and breast cancer.

Currently, phytoestrogens, which exhibit weak estrogenic activity, are very active in the prevention of postmenopausal osteoporosis (Aldercreutz H, Mazur W., Proc Nutr Soc., 55: 399, 1996). For example, genistein and diadzein among isoflavones, which are contained in a large amount of soybean, are estrogen-like active substances, and osteoblast-like cells or osteoblasts of soybean ethanol extract and oxidative stress and free radicals and free radical scavenging activity (Lee YS et al al ., Korea Soybean Digest. , 18: 35,2001; Choi EM et al ., Phytochemistry , 56: 733, 2001; Choi EM et al ., Food Research International , 35: 893,2002). However, this method has limitations in that it is limited to causes other than menopausal osteoporosis.

In addition, calcium supplementation such as calcium salt, bovine bone meal, and vitamin D metabolite therapy have been used for the treatment of non-menopausal osteoporosis (Orcel P, Krane SM, Secondary osteoporosis and glucocorticoid include osteoporosis. Ann Med Interne. 2000), they have problems in terms of solubility, water absorption, and taste. (Karen MP, Carol CP, and Lawrence GR, Treatment of osteoporosis, Annu Rev Med. 6: 249-256 (1995)), but they are also generalized in terms of cost and side effects . On the other hand, proper exercises can increase bone mass and strengthen bones, but there are problems that it is difficult to exercise such as weak patients, candidates who are in short of exercise time, and elderly people with difficulty in movement. Bisphosphonates and calcitonin preparations have been used for the treatment of osteoporosis. However, these medicines are special medicines and have side effects such as being stuck in the esophagus. Therefore, , Headache, anorexia and other side effects.

On the other hand, it is a perennial herb that belongs to the Asteraceae which grows wild in the sandy soil of riverside or riverside in Korea. It does not die in the winter, but it is called "japonica" or "japonica" because it shoots again from the stem the next year.

It is also known to contain essential oil, coumarin, chromone, flavonoids, caffeic acid, aromatic oxycarbonic acid, various minerals and vitamins. And has been used for the treatment and prevention of various diseases such as liver function protection, antiviral, blood pressure strengthening, vasodilatory action, analgesic and antipyretic action, cholecystitis, jaundice, genomes, indigestion, menstrual disorders and pregnancy addiction. The major pharmacological components of the black mugwort are scoparone (6,7-dimethoxycumarine) and betaine. Scoparone is a coumarin derivative. It is present on the surface part of the black mugwort, , Is used as diuretic and has been reported to have high antioxidant activity, and is known to be effective for liver diseases in particular.

In particular, scoparone (6,7-dimethoxycoumarin), which is a coumarin derivative present in the black mugwort, has a structure in which benzene and pyrone-ring are combined, and has immunosuppression, vasorelaxation, It has been reported that various effects such as blood pressure lowering action, dandruff action, and anti-inflammatory action are reported (Korean Patent No. 1169217; Korean Patent No. 1093930). It has been reported in the past that osteoporosis is effective for the prevention and treatment of osteoporosis in osteoporosis under oxidative stress (Korean Patent Laid-Open Publication No. 2013-0091160), the prevention and treatment of osteoporosis .

Accordingly, the present inventors have made intensive efforts to develop a natural extract-derived substance free from side effects and effective for the prevention or treatment of osteoporosis. As a result, it has been found that scoparone increases osteoblast activity, inhibits osteoclast activity, The present inventors confirmed that the extracts containing osteoporosis derived osteoporosis were effective for the prevention and treatment of osteoporosis, and confirmed the effect of preventing or treating osteoporosis of the extracts.

It is an object of the present invention to provide an extract containing scopolamine derived from mackerel, which has excellent effects such as an increase in osteoblast cell activity, inhibition of osteoclast activity and inhibition of bone resorption activity, and a composition for preventing or treating osteoporosis comprising the same.

In order to achieve the above object, the present invention provides a composition for preventing or treating osteoporosis comprising scoparone represented by the following general formula (1) or the skeleton-containing hazelnut extract as an active ingredient, and the composition.

[Chemical Formula 1]

The present invention also provides a health functional food for preventing or ameliorating osteoporosis, which comprises scoparone represented by the general formula (1) or the skarfone-containing sea squirt extract as an active ingredient.

The scoparone of the present invention or the scarfolone-containing hazelnut extract of the present invention increases the osteoblast activity, inhibits osteoclast activity and inhibits bone resorption activity, and thus can be used not only for prevention and treatment of osteoporosis but also for improving and improving bone health, , A health functional food or a drink.

Fig. 1 is a schematic diagram showing the separation and purification of Rhodiola var.
FIG. 2 is a diagram showing the chemical formula for 14 kinds of substances separated from the black mugwort.
1: (+) - pinoresinol, 2: scopoletin, 3: ethanone, 4: 5-epi-yudesma-4 (15) 5-epi-eudesma-4 (15) -ene-1? -6? -Diol, 5: scoparone, 6: ficusic acid, 7: D-3-methoxy- 3-methoxy-4-hydroxy-phenylglycol, 8: umbelliferone, 9: 5,4'-dihydroxy-3,7-dimethoxy flavone (5, 9-ene-4,6-diene-1,8-diol 1-O-? -D-glutopyranoside (deca-9-en- 12: cyclopentanone, 13: 7S, 7'S, 8R, 8 ', 8'-dibenzothiophene, R-icariol A2-9-O-? -D-glucopyranoside, 14: tuberonic acid (7S, 7'S, 8R, acid.
Fig. 3 is data showing the effect of osteoblast proliferation of 14 different materials derived from rhizome.
FIG. 4 is a graph showing the effect of 14 kinds of substances derived from Japanese black mugwort on osteoblast ALP (alkaline phophatase) activity.
FIG. 5 is a graph showing the effect of 14 kinds of materials derived from Japanese black mulberry on the synthesis of osteoblastic collagen.
FIG. 6 is a graph showing the effect of 14 kinds of materials derived from Japanese black mugwort on osteoblast bone nodule formation (mineralization).
FIG. 7 is a graph showing the effect of 14 different materials from osteoclast on osteoclast differentiation activity through staining (top) and activity measurement (below) of the TRAP expression level of osteoclasts.
8 is data showing the effect of promoting osteoblast proliferation of scopolone.
9 is data showing the effect of scopolone on the ALP activity of osteoblast cells.
10 is data showing the effect of scopolar on osteoblastic collagen synthesis.
11 is data showing the effect of scopolone on osteoblast bone nodule formation (mineralization).
12 is data showing the degree of toxicity of scopolar extract to osteoclasts.
13 is data showing osteoclast TRAP staining and osteoclast counting effect on the osteoclast TRAP activity inhibition effect of scopolar.
14 is data showing the effect of scopolone on the osteoclast TRAP activity.
Fig. 15 is data showing bone resorption inhibitory effect of osteoclast differentiation of scopolar.
FIG. 16 shows the scopolar effect on ROS (A), O ₂ (B), H ₂ O ₂ (C) and GSH (D) levels during osteoclast differentiation.
17 is data showing the effect of scopolone on MMP (mitochondrial membrane potential) during osteoclast differentiation.
18 is data showing the effect of scopolone on the TRAF6 / Rac1 / Nox1 / ROS signaling pathway.
Figure 19 is data showing the effect of scopolar on c-Src and PI3K expression and activity, the up-regulation mediators of Rac1.
Figure 20 is data showing the effect of scopolone on the expression of antioxidant enzymes (Catalase and SOD1).
21 is data showing the scopolar effect on the phosphorylation of JNK, ERK and p38 which are signal pathway proteins of MAPK (Mitogen-Activated Protein Kinase).
22 is data showing the effect of controlling the expression of c-Fos and NFATc1 of scopolar.
Figure 23 is data showing the effect of scopolone on osteoclast markers Cathepsin K and TRA expression.
24 is a schematic view showing a production method of a skarfone-containing sea squirt extract.
25 is data showing the effect of the Sopomonospora japonica extract on body weight and dietary efficiency of experimental animals.
Fig. 26 shows data on the effect of the scarf-containing sea squirt extract on the kidney, liver and uterus of an experimental animal.
FIG. 27 shows data showing the effect of the skarfone-containing sea squirt extract on BMC (A) and BMD (B) of an experimental animal.
FIG. 28 shows data on the effect of scarfolone-containing sea squirt extract on PICP (procollagen type I C-terminal peptide) content in the blood of an experimental animal.
FIG. 29 shows data on the effect of scarfolone-containing sea squirt extract on BALP (Bone Alkaline Phosphatase) content in blood of an experimental animal.
FIG. 30 shows data showing the effect of the scarfolone-containing sea squirt extract on blood OC (osteocalcin) content of experimental animals.
FIG. 31 shows data on the effect of the scarfolone-containing sea squirt extract on the blood OPG (osteoprotegerin) content in blood of an experimental animal.
FIG. 32 shows data on the effect of the scarfolone-containing sea squirt extract on the serum RANKL (receptor activator of NF-κB ligand) in the experimental animals.
FIG. 33 shows data on the effect of scarfolone-containing sea squirt extract on serum TRAP (tartrate resistant acid phosphatase) content in blood of an experimental animal.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to a composition comprising scoparone represented by formula (1) and artemisia containing scopolone capillaris ) extract as an active ingredient for the prevention or treatment of osteoporosis.

[Chemical Formula 1]

In the present invention, the content of scopolone may be 0.001 to 20% by weight.

In one embodiment of the present invention, to confirm the effect of preventing or treating osteoporosis of each component contained in the black mugwort extract, seaweed component was separated and purified. As shown in Table 1 and FIG. 2, As a result of the analysis, it was confirmed that 1: (+) - pinoresinol, 2: scopoletin, 3: ethanone, 4: 5-epi-yudesma- 5-epi-eudesma-4 (15) -ene-1? -6? -Diol, 5: scoparone, 6: ficusic acid, 7: Methoxy-4-hydroxy-phenylglycol, 8: umbelliferone, 9: 5,4'-dihydroxy-3,7-dimethoxy Dihydroxy-3,7-dimethoxyflavone, 10: deca-9-ene-4,6-diyne-1,8-diol 1-O- beta -D-glutopyranoside (deca- 12-cyclopentanone, 13: 7S, 7 ' S, 9-en- 8R, 8 ' R- O-beta-D-glucopyranoside), 14: tuberonic acid (7S, 7'S, 8R, 8'R-icariol A2-9-0- .

To confirm osteoporosis prevention or treatment efficacy of the 14 substances derived from the above-mentioned Satsuma mugwort, osteoclast proliferation promoting effect and cytotoxicity (Fig. 3), ALP (alkaline phophatase) activity (Fig. 4), collagen synthesis ability ), Bone module formation (FIG. 6), and osteoclast TRAP activity (FIG. 7). As a result, In the present invention, scoparone was selected from among 14 substances derived from mulberry, and the effect of scopolar derived from Sophora spp. On the prevention or treatment of osteoporosis was examined.

Osteoporosis is a disease caused by excessive bone resorption rather than osteoporosis. It occurs when osteoblast activity is weakened and osteogenesis is decreased, osteoclast activity is strengthened, bone resorption is increased, Therefore, in order to confirm osteoporosis prevention and therapeutic effect, the osteoblast activation and the inhibition of osteoclast differentiation were measured in the present invention.

In order to investigate the effect of scopolone on the osteoblast activity, MC3T3-E1 subclone 4 cells were used to measure the proliferation of osteoblasts, ALP activity, collagen synthesis and bone nodule, Mineralization was confirmed. The cells were treated for 1, 10, and 20 μM concentration of scopolone and cultured for 6 days and 15 days. Cell proliferation and cytotoxicity were observed. As a result, cell proliferation activity was slightly observed, and cytotoxicity was not observed during the culture period of 15 days (Fig. 8). In the case of osteoblast ALP activity, 15.62 units, 16.44 units, and 18.19 units of scopolone treated with 1, 10, and 20 μM concentrations, respectively, increased ALP activity in a concentration-dependent manner. 17β-estradiol, used as a positive control, It was confirmed that the ALP activity was higher than the treated cells (15.60 units on the 6th day of differentiation) (Fig. 9).

In the case of osteoblast collagen synthesis, collagen synthesis was increased to 163.43 to 181.85%, and collagen synthesis effect similar to E2 (173.04%) used as a positive control was observed when scopolar was treated at concentrations of 1, 10 and 20 μM (Fig. 10). According to previous studies, I-type collagen produced from osteoblasts accounted for 85-90% of total bone protein (Noriyoshi K. Endocrinology. 1986), which suggests that scopolar promotes collagen synthesis, I can confirm that I can influence it.

The formation of bone consists of three stages of substrate proliferation, maturation and calcification (Pittenger JJ et al . Science., 1999), and the ability of bone nodule formation is known to be an important marker for osteoblast differentiation. Alizarin is a vegetable dye that is specifically adsorbed to calcium, which is known to be proportional to the amount of mineralization and staining, because it is stained to extracellular matrix of mineralized cells (Schiller PC, et al ., Bone., 2001).

In the present invention, treatment with scopolone at concentrations of 1, 10 and 20 μM resulted in an increase in the synthesis of bone nodules from 141.56 to 154.01% (E2; 157.84%) similar to the positive control group (FIG. 11) It is believed that PALON promotes extracellular matrix formation of osteoblast cells and positively affects osteogenesis.

In order to examine the effect of scopolarone on osteoclast differentiation activity, scopolar was treated with RAW 264.7 cells at a concentration of 1, 2, 5, 10 and 20 μM for 5 days, , Indicating no cytotoxicity at all concentrations (Fig. 12). As a result of confirming the TRAP-positive polynuclear osteoclast cells treated with scopolone at a concentration of 1 to 20 μM, the osteoclast formation was reduced dependently on the scopolonal concentration, and the number of osteoclasts was counted as 61.69 (Fig. 13). As a result of measuring TRAP activity as an osteoclast differentiation marker, it was confirmed that TRAP activity was decreased to 196.70 to 254.70% depending on the concentration of scopolone (Fig. 14). That is, it was confirmed that scopolar inhibits osteoclastogenesis and TRAP activity and inhibits reabsorption of bone, thereby preventing and treating osteoporosis.

Further, as a result of measuring the bone resorption activity by osteoclast differentiation, it was confirmed that the bone resorption activity was decreased from 192.76 to 228.64% depending on the concentration of scopolone (Fig. 15). It can be seen that scopolarone inhibits osteoclast differentiation and regulates bone metabolism by inhibiting bone resorption activity.

(ROS) and glutathione (GSH) are known to regulate osteoclast differentiation (Leper), and it is known that osteoclast-induced ROS, O ₂ , H ₂ O ₂ And GSH levels were measured using DCFH-DA, DHE, DHR123 and mBCL fluorescent probes. As a result, it was confirmed that the ROS and O ₂ levels increased by RANKL treatment were decreased in dependence on the scopolar concentration. ₂ O ₂ And the GSH level (Fig. 16). During osteoclast differentiation, ROS, especially O ₂ Generation is an important signal transducer in osteoclast differentiation, and scopolone can suppress osteoclast differentiation by regulating ROS (O ₂ ) generation.

The changes of mitochondrial membrane potential (MMP) in osteoclasts by scoparone were measured by Rhodamine 123 (rhodamine 123) fluorescence probe. The osteoclast MMP decreased by 34.94% compared with the control group during differentiation induction period, , It was confirmed that MMP increased in a concentration-dependent manner from 35.59 to 54.47% (FIG. 17).

These results suggest that ROS generation in the osteoclast differentiation process occurs in the mitochondria and scopolar inhibits osteoclast differentiation by regulating ROS generation by inhibiting the decrease of MMP by RANKL treatment.

In order to elucidate the mechanism of inhibition of osteoclast activation by scoparone in the present invention, the degree of osteoclast differentiation signal-related protein expression and activity were measured. RAW 264.7 cells were treated with scfalone at 0, 1, 10, and 20 μM concentrations with 50 ng / ㎖ receptor activator of nuclear factor-κB ligand (RANKL) for 2 days, And used for confirmation of protein expression.

In the osteoclast differentiation process, RANKL treatment increased intracellular ROS production through TRAF6 and NADPH oxidase (NADPH oxidase) signal transduction system, and it acts as a secondary important signal transducer in osteoclast differentiation . The absence of TRAF6 and the expression of the dominant mutant form inhibit the activity of Rac1, one of the components of the NADNADPH oxidase enzyme, indicating that Rac1 is an important mediator of osteoclast differentiation that functions in the sub-mechanism of TRAF6 (Lee et al ., 2005).

In the present invention, western blotting was performed to examine the effect of scopolarone on the regulation of ROS signaling molecules associated with TRAF6 / Rac1 / Nox1 in ranch-derived osteoclast differentiation, SCOPHARONE has been shown to decrease the expression of TRAF6, the highest regulator of the RANKL / RANK signaling pathway, in a dose-dependent manner and to decrease the activity of Rac1, a sub-mechanism mediator of TRAF6 (Fig. 18). These results indicate that Scarfone regulates the expression of TRAF6 and reduces the expression of GTP-Rac1, the active form of Rac1, the cytoplasmic subunit of NADPH oxidase, and prevents translocation of Rac1 to the cell membrane And inhibit the formation of NADPH oxidase complexes, thereby regulating the development of ROS.

Src, one of the family of c-Src is sikimyeo phosphorylation of p47 ^phox, one of the cytoplasmic subunit (cytoplasmic subunit) of the NADPH oxidase, phosphorylation of p47 ^phox is to facilitate the binding to membrane oxidation element (membrane oxidases componet), PI3K and Rac1 (Cai H. et < RTI ID = 0.0 > al . Trends in Pharm, 2003). In addition, it has been reported that the signal transduction system via osteoclast TRAF6 / c-Src / PI3K regulates osteoclast survival time through cytoskeletal reorganization and anti-apoptosis (Steven L, et al . Nat. Rev. Genet, 2003).

This suggests that the c-Src and PI3K signaling pathways are important mediators involved in the activation of Rac1 in the regulation of ROS signaling molecules associated with TRAF6 / Rac1 / Nox1 as well as the regulation of osteoclast survival, The expression levels of c-Src and PI3K were determined by Western blotting. As a result, the expression of c-Src was decreased dependently on the concentration of scopolone, Lt; RTI ID = 0.0 > PI3K < / RTI > (FIG. 19). These results suggest that scopolarone reduces the expression of c-Src and inhibits phosphorylation of PI3K, thereby regulating the activity of Rac1.

Oxidative stress caused by excessive in vivo reactive oxygen species (ROS) can be defined as a biological phenomenon that occurs when the balance between antioxidants and oxidants in cells or tissues is broken. Antioxidant enzymes present in the body, such as catalase, glutathione (GSH), glutathione peroxidase (GPX) and superoxide dismutase-1 (SOD-1)

In order to investigate the effect of scopolar on the expression of antioxidant enzymes (catalase and SOD1) in osteoclasts in relation to ROS generated through the RANKL / RANK signaling system, Western blotting was carried out, (Fig. 20), the expression of catalase and SOD1 decreased by treatment (Fig. 20). Scoparone was found to increase the expression of antioxidant enzymes (catalase and SOD1) And regulate osteoclast ROS generation through signal transduction pathways.

The mitogen-activated protein kinase (MAPK) pathway is known to be activated by a series of stimuli such as stress, heat shock, and radiation to induce various biological responses such as inflammation, apoptosis, and cell differentiation Ono K, et al . Cell signal, 2000). In osteoclasts, the binding of RANKL to RANK promotes TRAF family protein binding, and the RANKL signal is transmitted to the osteoclasts, such as JNK (c-Jun N-terminal kinase), p38, and extracellular signal-regulated kinases (ERK) (NF-kB), NFATc1, and c-Fos, which are essential for osteoclast differentiation, are known to regulate osteoclast differentiation and maintenance in osteoclast precursors (Galibert L, et al . J Biol Chem. 1998).

To examine the effect of scopolar on the expression of JNK, ERK and p38, which are activated through the RANKL / RANK signaling system, and on the lower signaling molecules c-Fos and NFATc1, Sukofarone was transfected with RANKL / TRAF6 / Rac1 / Nox signaling pathway (Fig. 21), it was confirmed that the expression of c-Fos, which is regulated by activation of JNK and ERK, And regulate the expression of NFATc1, which is regulated by them (Fig. 22).

Bone homeostasis is the process by which osteoblasts, which induce mineral accumulation, and osteoclasts, which break down proteinaceous enzymes and bone components, (Boisvert CA. et al. , Nature, 2005). TRAP and cathepsin K secreted from osteoclasts are known to be proteolytic enzymes secreted from osteoclasts. Particularly, cathepsin K is an important cysteine protease selectively expressed only in osteoclast, and bone constituents break down proteins and minerals to destroy bone (Kim JM et al. , Int J Mol Med, 2007).

Western blotting was carried out to examine the effect of scopolone on the enzymes directly affecting bone resorption, cathepsin K and TRAP. As a result, the expression of cathepsin K and TRAP secreted from osteoclast by scopolone was reduced (Fig. 23).

In conclusion, the inhibition of bone resorption by scopolone decreases the ROS that occurs during osteoclast differentiation through signal pathway to TRAF6 / Rac1 / Nox1, and also induces stress, heat shock, radiation It has been demonstrated that scopolarone is effective in the prevention and treatment of osteoporosis because it inhibits the activation of MAPKs pathway which is sensitive to a series of stimuli (ROS) and inhibits osteoclast-induced bone resorption.

The present invention relates to a method for producing Sopomus sinropodiflora extract of Skoparone or Scoparone, which comprises extracting Sophora sp. Extract with an extraction solvent containing 50% (v / v) syrup, (A) finely finely dividing the sample of rhizome, adding a first extraction solvent containing 50% alcohol (v / v), and heating the mixture to 55 to 65 ° C for primary extraction; (b) recovering and concentrating the primary extract, adding a secondary extraction solvent containing 50% (v / v) vat, and heating to 45 to 55 캜 for secondary extraction; And (c) recovering said secondary extract, followed by concentration and drying to obtain a skarfone-containing muskmelon extract (FIG. 24).

In the step (a), the first extraction solvent is added so as to be in a ratio of 1: 7 to 1: 9 with the rhizome sample, and the extraction is performed for 4 to 8 hours. In step (b) The extraction solvent is added so as to be in a ratio of 1: 4 to 1: 6 with the rhizome sample, and then extracted for 4 to 18 hours. The skim milk extract obtained in the step (c) contains 5.0 to 7.0 mg / g of scoparone.

In one embodiment of the present invention, the rosewater samples purchased from Yeongcheon, Gyeongbuk Province were divided into 5 pieces (4 kg) and finely divided. After that, 1 part of a mixture containing 50% alcohol (v / v) 32 ℓ of tea extracting solvent was added and the mixture was extracted at 60 ± 2 ° C. for 6 hours to recover the first extract. 20 L of a second extraction solvent containing 50% alcohol (v / v) so as to be a ratio of 1: 5 to the remaining sea tangle was added and extracted at 50 ± 2 ° C. for 16 hours to recover the second extract . The primary extract and the secondary extract were collected by vacuum or reduced pressure and concentrated. The concentrated primary extract and secondary extract were mixed and lyophilized or spray-dried to obtain a scarf-containing black mugwort extract. Each extract was frozen The concentration of the extract concentrate was measured by drying and the average yield was 13.8% (Table 2).

The results of HPLC analysis (Table 3 and Table 4) of the content of scopolone contained in the black mugwort extracts extracted by the method of the present invention and the control group contained about 0.12% of scopolone, It was confirmed that the extracts of five kinds of silkworm extract contained 0.54 to 0.67% of scopolone on average.

8-week-old female Sprague-Dawley (SD) rats were administered ovariectomized and sham operation in order to confirm osteoporosis prevention and treatment effects of the scarfolone-containing morning wolfberry extract isolated by the method of the present invention Were divided into five groups (n = 12 per group) as shown in Table 5, and 50 μg / kg of 17β-estradiol (E ₂ ) was given as a positive control group while calcium-deficient diets were fed to the experimental animals for a total of 84 days 125 mg / kg and 250 mg / kg of sea squirt extract were orally administered daily for 12 weeks.

(OVA + E2 50 ㎍ / ㎏), Group Ⅳ (OVA + ACE 125 ㎎ / ㎏) and Group Ⅴ (OVA + E2) OVA + ACE 250 ㎎ / ㎏). However, in the case of body weight, weight gain was increased by increasing fat accumulation by ovariectomy. However, it was confirmed that there was no toxicity and ingestion rejection by the black mugwort extract and did not affect the nutritional metabolism of the experimental animals (Fig. 25).

The kidney, liver, and uterus were extracted and analyzed at the end of the experiment (12 weeks) to confirm the effect of the scarfolone-containing mudberry extract on the kidney, liver, uterus and femoral bones of the experimental animals (BMC, BMD) , And left and right femoral bones. No significant difference was found in the height of kidneys and liver between groups. In the case of uterine weight, the weight of uterus was slightly increased in group Ⅲ positive, (Group Ⅳ and group Ⅴ) treated with the extracts of black mugwort (Fig. 26).

The weight, length and thickness of the thigh bone were not significantly different between groups Ⅱ and Ⅲ, Ⅳ and Ⅴ, but bone mineral density and mineral content of femur bone were significantly different between group II (OVA) and group I (OVA + ACE 125 mg / ㎏, Ⅴ: OVA + ACE 250 mg / kg) compared with the control group (OVA + E2 50 ㎍ / 27).

The results showed that 17β-estradiol used as a positive control (Ⅲ: Ova + E2 50μg / kg) decreased uterine regression due to ovariectomy. However, There was no significant difference in the weight of uterus in the group treated with extracts of urticaria (IV: OVA + ACE 125 ㎎ / ㎏, Ⅴ: OVA + ACE 250 ㎎ / ㎏) In addition, bone mineral density and mineral content of ovariectomized rats recovered to ovariectomized rats when they were orally administered to ovariectomized rats for 12 weeks, suggesting that scarfolone-containing sea urchin extracts are effective in the prevention and treatment of osteoporosis in menopausal women. It will be possible to contribute.

PICP (procollagen type Ⅰ C-terminal peptide) is produced in the osteoblast after being secreted to the precursor procollagen and then formed by proteolytic cleavage to become collagen. It can be used as a marker (Calvo. MS et al . Endocr Rev., 1996). In order to confirm the PICP regulatory effect of Sophora japonica extract containing Scoparone, PICP in the blood of the experimental animals was compared with that of Group Ⅲ (OVA + E2 50μg / ㎏) The PICP level of OVA + ACE 250 mg / kg was significantly increased (Fig. 28), and the administration of the squamopone-containing black mugwort extract increased type I collagen formation And it was confirmed that it can help bone formation.

BALP of BALP ( Bone Alkaline Phosphatase) in ovariectomized extracts containing Scoferon was measured. BALP of ovariectomized group (OVA + E2 50μg / ㎏) was compared with group Ⅱ (OVA) , BALP activity was significantly increased in group IV (OVA + ACE 125 mg / kg) and group V (OVA + ACE 250 mg / kg) (FIG. 29). BALP is a bone-specific enzyme secreted during osteoblastic differentiation and is involved in bone remodeling. Increased secretion of BALP by skarfone-containing mugwort extract promotes bone resorption and promotes osteoporosis prevention And can contribute to the treatment.

Osteocalcin (OC) is formed in osteoblasts and is deposited in bone matrix, which acts as a bone resorption signal of osteoclasts. Osteocalcin, known to be involved in the mineralization of bone, is known as bone formation because some of the new formation is released into the blood (Calvo. MS et al . Endocr Rev., 1996). The osteocalcin (OC) concentration in the ovariectomized group Ⅱ (OVA) was measured to determine the osteocalcin-modulating effect of the osteoporosis extract containing scoparone. As a result, the osteocalcin level increased in the ovariectomized group Ⅱ (OVA) (OVA + ACE 125 mg / kg) and group V (OVA + ACE 250 mg / kg) (Fig. 30). This is a decrease in the bone turnover caused by ovariectomy by the extraction of skarfone-containing snow pea extract.

OPG (osteoprotegerin) is a soluble decoy receptor that binds to RANKL and has the effect of blocking the RANKL signal and thus inhibiting the differentiation into mature osteoclasts. (OVA + E2 50 ㎍ / ㎏) and OVA + ACE (OVA + E2) group, which were compared with OVA group, were compared with ovariectomized group Ⅱ (OVA) 250 mg / kg), the secretion of OPG was increased (FIG. 31). In other words, the extract of Skarfarone, which contains opium, increases secretion of OPG to block RANK / RANKL signals and inhibits differentiation into mature osteoclasts, thereby reducing osteoclast-induced bone resorption, thereby reducing bone loss Respectively.

RANKL (receptor activator of NF-κB ligand) binds to RANK to induce mature osteoclast differentiation from hematopoietic stem cells and increase the survival time of mature osteoclasts. Therefore, Serum RANKL levels of the experimental animals were compared with those of ovariectomized group Ⅱ (OVA), group Ⅲ (OVA + E2 50 ㎍ / ㎏) and group Ⅳ (OVA + ACE 125 ㎎ / / Kg) and group V (OVA + ACE 250 mg / kg) significantly reduced RANKL production (FIG. 32). It was confirmed that the reduction of RANKL production by the scarfolone-containing sea squirt extract inhibits the RANK / RANKL signal to inhibit the differentiation into mature osteoclasts, thereby reducing osteoclast-induced bone resorption, thereby preventing and treating osteoporosis I could.

In order to investigate TRAP (tartrate resistant acid phosphatase) modulating effect of Sophora japonica extract, TRAP levels in ovariectomized group Ⅱ (OVA) were increased, but the positive control group (OVA + ACE 125 mg / kg) and group Ⅴ (OVA + ACE 250 mg / kg) were significantly higher than those of group Ⅱ (Fig. 33). That is, it was confirmed that the extract of Sophora japonica containing scoparone reduces the osteoclast-induced osteoclast-induced TRAP secretion, thereby preventing and treating osteoporosis.

The cross-linked C-terminal telopeptide of type I collagen (ICTP) is a type of collagen that binds to the osteoclast through the osteoclast, meat al . Calcif Tissue Int., 1998), ICTP concentration in the serum of the ovariectomized group Ⅱ (OVA) compared to group I (Sham) (OVA + E2 50 ㎍ / ㎏) and OVA + ACE 250 ㎎ / ㎏ (Ⅴ group), which is a group of ovariectomized extract containing scopolone, increased ICTP level compared to group Ⅱ (OVA) (Fig. 33).

That is, the skarfalone-containing black mugwort extract prepared by the method of the present invention has an effect of increasing the bone mineral density and mineral content of the bone, promoting the formation of type I collagen (type I collagen) and BALP secretion to promote osteoporosis And to help with the treatment. In addition, it has an effect of reducing bone turnover caused by ovariectomy, and it inhibits OPG secretion, RANK / RANKL signal, TRAP and ICTP, inhibits differentiation into mature osteoclasts, , The composition for enhancing bone dryness containing skeleton derived skeleton of the present invention as an active ingredient is effective for promoting osteoblast activity and inhibiting bone resorption of osteoclasts to prevent and treat osteoporosis Respectively.

The composition for preventing and treating osteoporosis of the present invention may be formulated together with the medicines already used or may be used in combination. The pharmaceutical dosage forms of the compositions of the present invention may also be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutical mg / kg active compounds as well as in a suitable set.

The composition for preventing and treating osteoporosis, which contains, as an active ingredient, Scooparone or Scoparone-containing sea squirt extract according to the present invention, can be administered orally or parenterally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, And may be formulated in the form of a tablet, an external preparation, a suppository, and a sterile injection solution.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at a daily dose of 0.001 to 200 mg / kg, preferably 0.01 to 100 mg / kg. The administration may be carried out once a day, or in several divided doses orally. The dose is not intended to limit the scope of the invention in any way.

The extract of the present invention can be administered to mammals such as rats, mice, livestock, humans and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

In another aspect, the present invention relates to a health functional food for improving osteoporosis prevention comprising scopolar of formula (1) or a skarfone-containing sea squirt extract as an active ingredient.

[Chemical Formula 1]

In the present invention, the content of scoparone or the skarfone-containing sea squirt extract contained in the health functional food for osteoporosis prevention and improvement may be 0.001 to 20% by weight.

In the present invention, the term "health functional food" means a food having improved functionality of a general food by adding the extract of the present invention to the general food. When the extract of the present invention is added to a general food, the physical properties and physiological functions of the general food will be improved, and the present invention can be broadly classified into 'health function Food ".

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such an additive is not so important, but is generally selected in the range of 0.01 to 20 parts by weight per 100 parts by weight of the extract of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Separation and purification of tea mugwort components and confirmation of bone health promotion effect

1-1: Separation of red mugwort

In order to examine the effects of each ingredient contained in the extracts on the osteoporosis, osteoporosis was investigated.

2.5 ㎏ of white wormwood collected from Yeongcheon, Gyeongbuk Province was extracted with 70% methanol (MeOH) three times to obtain 185 g of extract. The obtained methanol extract was suspended in distilled water (H ₂ O), and fractionated in turn using carbon tetrachloride (CH ₂ Cl ₂ ) and ethyl acetate (EtOAc) to obtain a carbon tetrachloride fraction, an ethyl acetate fraction and a distilled water fraction. The carbon tetrachloride fraction was subjected to silica gel column chromatography using a gradient solvent mixture of hexane: ethyl acetate (Hexane: EtOAc) at a ratio of 1: 0 to 0: 1 to obtain two fractions , Fr. 2), and Fr. 2 was subjected to Sephadex LH 20 column chromatography using a developing solvent in which acetone: distilled water (Acetone: H ₂ O) was mixed at a ratio of 1: 0 to 0: 1. 2A - Fr. 2C). Finally, Fr. Compounds 1, 2, 3, 4, 5, 6, 7, 8 were isolated and purified in the 2A fraction, Compound 9 was isolated and purified from the 2B fraction.

The distilled water fraction (140 g) was subjected to Diaion HP 20 column chromatography using a gradient solvent in which distilled water: methanol was mixed in a ratio of 1: 0 to 0: 1 to obtain five fractions (Fr 7 to Fr. 11), and Fr. Compounds 10, 11, 12, 13, and 14 were isolated and purified (Fig. 1).

Components derived from dead mugwort Sample No. Chemical name One (+) - Pinoleinol 2 Scopoletin 3 Ethanone 4 5-epi-eudesma4 (15) -ene-1.beta.-6 [beta] -diol 5 Scoparone 6 Ficusic acid 7 D-3-methoxy-4-hydroxyphenylglycol 8 Umbelliferone 9 5,4'-dihydroxy-3,7-dimethoxyflavone 10 Deca-9-ene-4,6-diyne-1,8-diol 1-O- [beta] -D-glucopyranoside 11 pumilaside 12 cyclopentanone 13 7S, 7S, 8R, 8R-icariol A2-9-O-b-D-glucopyranoside 14 tuberonic acid

2, scopoletin, 3: ethanone, 4: 1, 2, 3, 4, 5, 5-epi-eudesma-4 (15) -ene-1? -6? -Diol, 5: scoparone, 6 : Ficusic acid, D-3-methoxy-4-hydroxy-phenylglycol, 8: umbelliferone, 9: , 4'-dihydroxy-3,7-dimethoxyflavone, 10: deca-9-ene-4,6-diyne-1,8-diol 1 11-fumaraside, 12-o-ss-glutopyranoside, 10-o-ss-glutopyranoside, : Cyclopentanone, 13: 7S, 7'S, 8R, 8'R-icariol A2-9-0B-D-glutopyranoside (7S, 7'S, 8R, 8'R- 9-O-? -D-glucopyranoside) and 14: tuberonic acid.

To confirm osteoporosis prevention and treatment efficacy of the 14 kinds of materials derived from the above-mentioned puree mugwort, the osteoblast proliferation promoting effect, cytotoxicity, ALP (alkaline phophatase) activity, collagen synthesis ability, bone module formation and osteoclast activity degree were analyzed Respectively.

1-2: Promoting effect of osteoblast proliferation and cytotoxicity analysis of 14 substances derived from mulberry

MC3T3-E1 subclone 4 cells (osteoblast, ATCC, No. CRL-2593) were placed in a 24-well plate at a density of 5 × 10 ⁴ cells / well and incubated for 24 hours at 37 ° C. in a CO ₂ incubator pre-incubation). Then, 14 wells of 14 different materials were treated to 1 μM and 10 μM, respectively, and cultured for 48 hours. After 48 hours, the medium was removed and a solution of 5 mg / ml MTT (3- (4,5-dimethylthiazol) -2,5-diphenyltetrazolium bromide) was added to each well. The cells were incubated for 4 hours at 37 ° C in a CO ₂ incubator Lt; / RTI > After the MTT solution was removed, the cells were washed twice with 500 μl of PBS (pH 7.4) and treated with 300 μl of dimethyl sulfoxide (DMSO) to dissolve the cells. 200 쨉 l of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 570 nm with a microplate reader.

As a result, it was confirmed that all the substances except for 7, 8, and 9 of the 14 species of safflower treated with 1 μM and 10 μM significantly increased osteoblast proliferation (FIG. 3).

1-3: osteoblast ALP ( alkaline phophatase ) Effectiveness of 14 Substances from Rootstock

MC3T3-E1 subclone 4 cells were plated in a 24-well plate at 5 × 10 ⁴ cells / well, pre-incubated in a CO ₂ incubator at 37 ° C. for 24 hours, β-glycerolphosphate, 50 μg / ㎖ ascorbic acid) and cultured for 24 hours. The cells were cultured for 6 days in the presence of 14 different materials, and the cells were washed twice with 500 μl of PBS (pH 7.4), and 100 μl of 0.1% Triton X- 100 was added and lysed at 37 캜 for 30 minutes. The lysed cells were centrifuged at 14,000 rpm at 4 ° C for 10 minutes. 5 μl of the supernatant was used for protein quantification, and 30 μl of the supernatant was dissolved in 0.5M Tris-HCl buffer containing 1 mM MgCl ₂ and 6 mM p-nitrophenyl phosphate pH 9.8) was added to each well of a 96-well plate. After reacting at 37 ° C for 1 hour, 100 μl of 0.1 N NaOH was added to stop the reaction, and the absorbance was measured at 405 nm with an ELISA reader.

The alkaline phosphatase activity was determined by using 4-nitrophenol at 0, 10, 50, 100, 200 and 500 nM. The alkaline phosphatase activity was affected by the number of cells, The activity of phosphatase was calculated and the alkaline phosphatase activity was expressed as 1 unit (nM PNPP / mg of protein / min).

As a result, the MC3T3-E1 subclone 4 cells induced differentiation induction medium showed an increase of ALP activity up to 10.63 units compared to the cells not treated with differentiation inducing medium, and 2, 5 and 9 (Fig. 4). The results are shown in Fig.

1-4: Osteoblast collagen ( collagen ) Synthesis of 14 Sources from the Rootstock

MC3T3-E1 subclone 4 cells were plated in a 24-well plate at 5 × 10 ⁴ cells / well, pre-incubated in a CO ₂ incubator at 37 ° C. for 24 hours, β-glycerolphosphate, 50 μg / ml ascorbic acid) and cultured for 24 hours. After the medium was removed, the cells were washed twice with 500 μl of PBS (pH 7.4) and 500 μl of Bouin's fluid solution was added to each well. C for 1 hour, and then the cells were washed twice with 500 μl of PBS (pH 7.4). After that, the cells were stained with 0.1% Direct red 80 (Sirius red F3BA) solution for 1 hour. Then, unlabeled dye was removed using Acidic water, treated with 500 μl of 0.1 N NaOH solution, And the absorbance at 540 nm was measured with an ELISA reader.

As a result, the MC3T3-E1 subclone 4 cells induced differentiation induced differentiation induces collagen synthesis up to 131.47% compared to the cells not treated with differentiation inducing medium. As a result, it was confirmed that collagen synthesis was increased by most substances except 1, 4 and 9 (FIG. 5). These results suggest that most of the fractions obtained from the black mugwort can stimulate collagen production in osteoblasts and thus positively affect bone formation.

1-5: osteoblast Bone nodule ( bone nodule ) formation( mineralization Effects of 14 kinds of mulberry seeds

MC3T3-E1 subclone 4 cells were plated in a 24-well plate at 5 × 10 ⁴ cells / well, pre-incubated in a CO ₂ incubator at 37 ° C. for 24 hours, β-glycerolphosphate, 50 μg / ml ascorbic acid) and cultured for 24 hours. After the medium was removed, the cells were washed twice with 500 μl of PBS (pH 7.4), and the cells were washed with 500 μl of PBS (pH 7.4) Solution at 4 ° C for 1 hour, and then washed twice with 500 μl of distilled water. Thereafter, the cells were stained with 40 mM Alrizarin red S (pH 4.2) solution for 10 minutes, and then 500 증 of distilled water was used to remove undyed dye. 500 μl of 10% cetylpyridinium chloride was added to the plate, and 200 μl of the plate was transferred to a 96-well plate. Absorbance was measured at 562 nm using an ELISA reader.

As a result, in the MC3T3-E1 subclone 4 cells induced differentiation induction medium, the formation of bone nodule was increased up to 142.5% as compared with the cells not treated with differentiation inducing medium, and 14 kinds of cells Most of the substances except 1 and 13 were found to increase bone nodule formation compared to the differentiation inducing group (Fig. 6). Thus, 14 kinds of substances fractionated from red mugwort showed extracellular tempering of osteoblasts And bone formation.

1-6: osteoclast TRAP ( tartrate - resistant acid 포스화제 ) Effect of 14 Substances on the Dyeing and Activity

RAW 264.7 cells (osteoclast, ATCC, No. TIB-71) were placed in a 24-well plate at a density of 1 × 10 ⁵ cells / well and incubated for 24 hours at 37 ° C. in a CO ₂ incubator ) And treated with 14 different materials from rosewood with 5 μM of differentiation induction medium (50 ng / ㎖ receptor activator of nuclear factor-κB ligand (RANKL)). The cells were cultivated for 5 days in the presence of 14 different materials, and the cells were washed once with 500 μl of PBS (pH 7.4), and the cells were washed with 4% formaldehyde Cells were fixed by treatment with ethanol / acetone (1: 1) for 1 minute for 10 minutes. Then, TRAP staining was performed using Leukocyte Acid Phosphatase Kit 387-A (Sigma, USA) after washing twice with 500 μl of distilled water.

TRAP activity was determined by treating RAW 264.7 cells with ethanol / acetone as in the staining procedure, washing twice with distilled water, and eluting with a 50 mM sheet containing 10 mM sodium tartrate and 5 mM ρ-nitrophenylphosphate 500 쨉 l of citrate buffer (pH 4.5) was added to each well, followed by reaction at 37 째 C for 1 hour. After the reaction, 100 μl of the enzyme reaction solution was dispensed into a new plate, the reaction was stopped with 0.1N NaOH, and the absorbance was measured at 405 nm.

When 14 kinds of substances isolated from black mugwort were treated at a concentration of 5 μM, most of the substances except 4 and 12 substances significantly decreased TRAP activity compared to the differentiation inducing group (196.85%) 7).

In the present invention, scopolone substances having the effect of promoting the osteoblast activity and inhibiting the osteoclast activity were selected from among 14 substances separated from the black mugwort, and thereafter, the effect of the osteoblast-derived scoparone on the bone health Respectively.

Derivation of osteoblastic cells on osteoblast differentiation activity Scooparon's  effect

2-1: Scooparon's  Promoting osteoblast proliferation and cytotoxicity analysis

MC3T3-E1 subclone 4 cells were placed in a 24-well plate at 5 × 10 ⁴ cells / well and pre-incubated for 24 hours at 37 ° C. in a CO ₂ incubator. Then, , 0, 1, 10, and 20 μM, and then cultured for 6 days and 15 days. After the culture, the medium was removed and a solution of 5 mg / ml of MTT (3- (4,5-dimethylthiazol) -2,5-diphenyltetrazolium bromide) was added to each well and cultured in a CO ₂ incubator at 37 ° C. for 4 hours . After the MTT solution was removed, the cells were washed twice with 500 μl of PBS (pH 7.4) and treated with 300 μl of dimethyl sulfoxide (DMSO) to dissolve the cells. 200 쨉 l of the supernatant was transferred to a 96-well plate and absorbance was measured at 570 nm with a microplate reader (ELISA reader).

Sucofarone was treated at 1, 10, and 20 μM concentrations, and cultured for 6 days and 15 days. Cell proliferation and cytotoxicity were observed. As a result, cell proliferation activity was slightly observed, and cytotoxicity was not observed during the culture period of 15 days (Fig. 8). The scopolone concentration in the range of 1 to 20 [mu] M was set in the subsequent activity evaluation.

2-2: Osteoblast cells ALP  Active Scooparon's  Efficacy measurement

MC3T3-E1 subclone 4 cells (cell information) were added to a 24-well plate at 5 × 10 ⁴ cells / well and pre-incubated for 24 hours at 37 ° C. in a CO ₂ incubator. The medium was replaced with induction medium (10 mM β-glycerolphosphate, 50 μg / ml ascorbic acid) and cultured for 24 hours. Sucopharon was treated at concentrations of 0, 1, 10 and 20 μM. The cells were cultured for 3 days, 6 days, and 9 days after replacing the medium containing scopolone at intervals of 3 days. After the medium was removed, the cells were washed twice with 500 μl of PBS (pH 7.4) Triton X-100 was added and lysed at 37 占 폚 for 30 minutes. The lysed cells were centrifuged at 14,000 rpm at 4 ° C for 10 min. 5 μl of the supernatant was used for protein quantification, and 30 μl of the supernatant was suspended in 0.5 M Tris-HCl buffer containing 1 mM MgCl ₂ and 6 mM p-nitrophenyl phosphate pH 9.8) was added to each well of a 96-well plate. After reacting at 37 ° C for 1 hour, 100 μl of 0.1 N NaOH was added to stop the reaction, and the absorbance was measured at 405 nm with an ELISA reader. The alkaline phosphatase activity was determined by using 4-nitrophenol at 0, 10, 50, 100, 200 and 500 nM. The alkaline phosphatase activity was affected by the number of cells, The activity of phosphatase was calculated and the alkaline phosphatase activity was expressed as 1 unit (nM PNPP / mg of protein / min).

As a result, 17β-estradiol used as a positive control increased activity to 15.60unit at 6 days of differentiation compared with cells treated with differentiation inducing medium, and 15.62unit, 16.44unit of scopolone treated at 1, 10, and 20μM concentration, respectively unit and 18.19 units of ALP activity (FIG. 9). On the 9th day of differentiation, ALP activity tended to decrease in each group as compared to day 6, which was judged to be due to the formation of bone nodule. This suggests that scopolar induces osteoblast differentiation, And may be involved in cell calcification.

2-3: Synthesis of osteoblast collagen collagen synthesis ) On Scooparon's  effect

MC3T3-E1 subclone 4 cells were plated in a 24-well plate at 5 × 10 ⁴ cells / well, pre-incubated in a CO ₂ incubator at 37 ° C. for 24 hours, β-glycerolphosphate, 50 μg / ml ascorbic acid), cultured for 24 hours, and treated with scopolar at concentrations of 0, 1, 10 and 20 μM. Cells were washed twice with 500 μl of PBS (pH 7.4), and washed with 500 μl of Bouin's fluid at 37 ° C for 1 day. And the cells were washed twice with 500 μl of PBS (pH 7.4). After that, the cells were stained with 0.1% Direct red 80 (Sirius red F3BA) solution for 1 hour. Then, the unstained dye was removed using acidic water, treated with 500 μl of 0.1 N NaOH solution, And the absorbance at 540 nm was measured with an ELISA reader.

As a result, in MC3T3-E1 subclone 4 cells induced differentiation induction medium, collagen synthesis was increased up to 154.45% compared with cells not treated with differentiation inducing medium, and E3 used as a positive control was 173.04 % Collagen synthesis. As a result of treatment with scopolone at concentrations of 1, 10 and 20 μM, it was confirmed that the collagen synthesis was increased from 163.43 to 181.85% (FIG. 10).

2-4: osteoblast Bone nodule ( bone nodule ) formation( mineralization ) On Scopa The Effects of Ron

MC3T3-E1 subclone 4 cells were plated in a 24-well plate at 5 × 10 ⁴ cells / well, pre-incubated in a CO ₂ incubator at 37 ° C. for 24 hours, β-glycerolphosphate, 50 μg / ml ascorbic acid), cultured for 24 hours, and treated with scopolar at concentrations of 0, 1, 10 and 20 μM. Cells were washed twice with 500 μl of PBS (pH 7.4), and washed with 500 μl of 70% ethanol at 4 ° C for 1 day. For a period of time, and then the cells were washed twice with 500 mu l of distilled water. After staining with 40 mM Alrizarin red S (pH 4.2) solution for 10 minutes, the unstained dye was removed with 500 μl of distilled water and 500 μl of a 10% solution of cetylpyridinium chloride After dissolving, 200 μl was transferred to a 96-well plate and absorbance was measured at 562 nm with an ELISA reader.

As a result, MC3T3-E1 subclone 4 cells induced differentiation induction medium showed 121.74% increase in bone nodule formation compared to cells not treated with differentiation induction medium, and E2 used as a positive control 157.84% (Fig. 11). As a result of treatment with scopolone at concentrations of 1, 10 and 20 μM, the synthesis of bone nodules increased from 141.56 to 154.01%, indicating that scopolarin promoted extracellular matrix formation of osteoblasts and positively affected osteogenesis .

On osteoclast differentiation activity Scooparon's  Effects and mechanisms

3-1: Scofaron  Toxicity of osteoclasts

RAW 264.7 cells were placed in a 24-well plate at 1 × 10 ⁵ cells / well, pre-incubated in a CO ₂ incubator at 37 ° C. for 24 hours, 1, 2, 5, 10, and 20 μM, and cultured for 5 days. After the culture, the medium was removed and a solution of 5 mg / ml of MTT (3- (4,5-dimethylthiazol) -2,5-diphenyltetrazolium bromide) was added to each well and cultured in a CO ₂ incubator at 37 ° C. for 4 hours . After the MTT solution was removed, the cells were washed twice with 500 μl of PBS (pH 7.4) and treated with 300 μl of dimethyl sulfoxide (DMSO) to dissolve the cells. 200 쨉 l of the supernatant was transferred to a 96-well plate and absorbance was measured at 570 nm with a microplate reader (ELISA reader).

Sucofarone was treated to 1, 2, 5, 10, and 20 μM concentration, and cultured for 5 days to confirm cytotoxicity. As a result, cytotoxicity was not observed at all concentrations (FIG. 12) RTI ID = 0.0 > uM. ≪ / RTI >

3-2: Effect on osteoclast differentiation activity Scooparon's  Effect - osteoclast TRAP  Dyeing and osteoclast counting counting )

RAW 264.7 cells were placed in a 24-well plate at a concentration of 1 × 10 ⁵ cells / well and pre-incubated in a CO ₂ incubator at 37 ° C. for 24 hours. Then, the cells were cultured in a differentiation induction medium (50 ng / activator of nuclear factor-κB ligand (RANKL)) at concentrations of 0, 1, 10, and 20 μM. Cells were washed once with 500 μl of PBS (pH 7.4), and then incubated with 4% formaldehyde for 10 min, Cells were fixed by treatment with ethanol / acetone (1: 1) for 1 minute. Then, TRAP staining was performed using Leukocyte Acid Phosphatase Kit 387-A (Sigma, USA) after washing twice with 500 μl of distilled water.

After staining, TRAP-positive polynuclear cells with three or more nuclei were counted using an optical microscope and used as an index of osteoclastogenesis.

As a result of confirming TRAP-positive polynuclear osteoclasts, osteoclast formation was reduced in a dependent manner on scoparone concentration, and TRAP-positive polynuclear cells were counted to effectively inhibit osteoclast differentiation to 61.69 to 93.15% 13).

3-3: Effect on osteoclast differentiation activity Scooparon's  Effect - osteoclast TRAP  activation

RAW 264.7 cells were placed in a 24-well plate at a concentration of 1 × 10 ⁵ cells / well and pre-incubated in a CO ₂ incubator at 37 ° C. for 24 hours. Then, the cells were cultured in a differentiation induction medium (50 ng / activator of nuclear factor-κB ligand (RANKL)) at concentrations of 0, 1, 10, and 20 μM. Cells were washed once with 500 μl of PBS (pH 7.4), and then with 4% formaldehyde for 10 minutes. The cells were washed twice with PBS (pH 7.4) , And treated with ethanol / acetone (1: 1) for 1 minute to fix the cells. Then, after washing twice with 500 μl of distilled water, 500 μl of a 50 mM citrate buffer (pH 4.5) containing 10 mM sodium tartrate and 5 mM ρ-nitrophenylphosphate And then reacted at 37 ° C for 1 hour. After the reaction, 100 μl of the enzyme reaction solution was dispensed into a new plate, the reaction was stopped with 0.1N NaOH, and the absorbance was measured at 405 nm.

As a result of measuring TRAP activity as an osteoclast differentiation marker, it was confirmed that the TRAP activity was reduced to 196.70 to 254.70% depending on the concentration of scopolone (Fig. 14), and scopolar inhibited osteoclast formation and TRAP activity, By inhibiting absorption, it can have a positive effect on the prevention and treatment of osteoporosis.

3-4: Effect on osteoclastic activity by osteoclast Scooparon's  effect

The osteoclast-induced bone resorption activity was measured using the Cosmobio bone resorption assay kit (CSR-BRA-48KIT). 250 [mu] l of Fluoresceinamine-labeld chondroitin sulfate (FACS) was applied to a 48-well plate coated with calcium phosphate (CaP-coated plate) and labeled at 37 [deg.] C for 2 hours. After the FACS solution was removed, the cells were washed twice with PBS (pH 7.4), and then treated with phenol red free (1 mL) until RAW 264.7 cells were cultured.

RAW 264.7 cells were seeded onto the above-mentioned 48-well plate (FACS-labeled CaP-coated plate) at a density of 5 × 10 ³ cells / well and cultured in phenol red free medium for 24 hours at 37 ° C. , And pre-incubated in a CO ₂ incubator and treated with 0, 1, 10, and 20 μM concentrations of scopolone with differentiation induction medium (100 ng / ml receptor activator of nuclear factor-κB ligand (RANKL) . The culture medium was exchanged for 2 days with a scaffold-containing differentiation medium for a total of 6 days. 100 μl of the culture was transferred to a 96-well black plate, and 50 μl of bone resorption buffer assay buffer) was added to each well and fluorescence (Ex 485 nm / Em 535 nm) was measured.

As a result of measuring the bone resorption activity by osteoclast differentiation, it was confirmed that the bone resorption activity was decreased from 192.76 to 228.64% depending on the concentration of scopolone (Fig. 15). It can be seen that scopolarone inhibits osteoclast differentiation and regulates bone metabolism by inhibiting bone resorption activity.

3-5: osteoclast differentiation ROS , O ₂ , H ₂ O ₂  And GSH  Level for this Scofaron  effect

(ROS) and glutathione (GSH) are known to regulate osteoclast differentiation (Leper), and it is known that osteoclast-induced ROS, O ₂ , H ₂ O ₂ And GSH levels were measured using DCFH-DA, DHE, DHR123 and mBCL fluorescent probes.

RAW 264.7 cells were plated in a 96-well plate at a density of 1 × 10 ⁵ cells / well, preincubated for 24 hours at 37 ° C. in a CO ₂ incubator, and cultured in a differentiation induction medium (50 ng / activator of nuclear factor-κB ligand (RANKL)) at concentrations of 0, 1, 10, and 20 μM. After incubation for 2 days, the medium was removed and replaced with 200 μl of HBSS, and DCFH-DA (40 μM), DHE (50 μM), DHR123 (20 μM) and mBCL Min. ≪ / RTI > Fluorescence of DCFH-DA (Ex 485nm / Em 535nm), DHE (Ex 485nm / Em 590nm), DHR 123 (Ex 485nm / Em 535nm) and mBCL (Ex 380nm / Em 465nm) , Switzerland).

Osteoclast ROS, O ₂ , H ₂ O ₂ And GSH levels, it was confirmed that the ROS and O ₂ levels increased by RANKL treatment were decreased in dependence on the scopolar concentration, and it was confirmed that they did not affect the H ₂ O ₂ and GSH levels (FIG. 16).

3-6: osteoclast differentiation MMP ( Mitochondrial membrane potential ) For Scooparon's  effect

Changes in mitochondrial membrane potential (MMP) in osteoclasts by scopolone were measured using rhodamine 123 (rhodamine 123) fluorescent probe.

RAW 264.7 cells were plated in a 96-well plate at a density of 1 × 10 ⁵ cells / well, preincubated for 24 hours at 37 ° C. in a CO ₂ incubator, and cultured in a differentiation induction medium (50 ng / activator of nuclear factor-κB ligand (RANKL)) at concentrations of 0, 1, 10, and 20 μM. After 2 days of culture, the medium was removed and replaced with 200 μl of HBSS. Then, 5 μM of rhodamine 123 was further added thereto and cultured for 30 minutes. After removing the medium and washing the cells with 50 μl of HBSS, 200 μl of HBSS was added and fluorescence was measured at Ex 485 nm / Em 535 nm using a GENios fluorescence plate reader (TECAN trading AG, Switzerland) .

As a result, MMPs in osteoclasts were reduced by 34.94% during induction of differentiation compared to the control group, but MMP was increased in a concentration-dependent manner from 35.59 to 54.47% when treated with scopolone (FIG. 17).

Osteoclast differentiation signal ( signaling pathway ) ≪ / RTI > Scooparon's  effect

In order to confirm the effect of scopolone on the expression and activity of osteoclast differentiation signal-related proteins, the cells were inoculated into a 24-well plate of RAW 264.7 cells at 1 × 10 ⁵ cells / well, from ₂ incubator adhered (pre-incubation) Scoring paron with the following differentiation induction medium (50ng / ㎖ receptor activator of nuclear factor-κB ligand (RANKL)) is treated so that 0, 1, 10, 20μM concentration. The cells were incubated for 2 days. After the medium was removed, the cells were washed once with 500 μl of PBS (pH 7.4), and proteins were isolated using methods known in the art. Separated proteins were used in subsequent protein expression confirmation experiments.

4-1: TRAF6 / Rac1 / Nox1 / ROS  For the signaling pathway Scooparon's  effect

Western blotting was performed to examine the effect of scopolarone on RANKL-induced osteoclast differentiation by TRAF6 / Rac1 / Nox1-related ROS signaling molecules.

As a result, it was confirmed that scoparone decreased the expression of TRAF6, which is the highest regulator of the RANKL / RANK signal pathway, in a concentration-dependent manner and decreased the activity of Rac1, a sub-mechanism mediator of TRAF6 (FIG. 18). These results indicate that Scarfone regulates the expression of TRAF6 and reduces the expression of GTP-Rac1, the active form of Rac1, the cytoplasmic subunit of NADPH oxidase, and prevents translocation of Rac1 to the cell membrane And inhibit the formation of NADPH oxidase complexes, thereby regulating the development of ROS.

4-2: Rac1 Lt; RTI ID = 0.0 > c- Src  And PI3K  For expression and activity Scooparon's  effect

Western blotting was performed to examine the effect of scopolone on the expression and activity of c-Src and PI3K when treated with concentration.

As a result, it was confirmed that the expression of c-Src was decreased dependently on the scopolar concentration and it was confirmed that it inhibited phosphorylation of PI3K, which is known as a sub-mechanism of c-Src (Fig. 19). These results suggest that scopolarone reduces the expression of c-Src and inhibits phosphorylation of PI3K, thereby regulating the activity of Rac1.

4-3: Antioxidant enzyme ( Catalase ( Catalase ) And SOD1 ) For expression Scooparon's  effect

Western blot analysis was performed to examine the effect of scopolone on the expression of antioxidant enzymes (osteoclast catalase and SOD1) in osteoclasts in relation to ROS produced by the RANKL / RANK signaling system.

As a result, it was confirmed that the expression of catalase and SOD1 reduced by RANKL treatment by scoparone was increased (Fig. 20), and scoparone increased the expression of antioxidant enzymes (catalase and SOD1) It was confirmed that ROS generation in osteoclasts was regulated through two signal transduction pathways that cleared the generated ROS.

4-4: MAPK ( Mitogen - Activated Protein Kinase ) For the signal path Scofaron  effect

Western blotting was performed to examine the effect of scopolar on the expression of JNK, ERK, and p38, which are activated through the RANKL / RANK signaling system, and the lower signaling molecules c-Fos and NFATc1.

As a result, it was confirmed that scopolar inhibits phosphorylation of activated JNK, ERK and p38 through oxidative stress generated through the RANKL / TRAF6 / Rac1 / Nox signaling pathway (FIG. 21) To regulate the expression of NFATc1, which is regulated by them, (Fig. 22).

4-5: osteoclast Marker Inn Cathepsin  K ( Cathepsin  K) and TRA  For expression Scofaron Effect of

Western blotting was carried out to examine the effect of scopolone on the enzymes directly affecting bone resorption, cathepsin K and TRAP. As a result, the expression of cathepsin K and TRAP secreted from osteoclast by scopolone was reduced Respectively.

In conclusion, the inhibition of bone resorption by scopolone decreases the ROS that occurs during osteoclast differentiation through signal pathway to TRAF6 / Rac1 / Nox1, and also induces stress, heat shock, radiation It was confirmed that MAPKs pathway activity, which is sensitive to a series of stimuli (ROS), is inhibited and bone resorption by osteoclasts is inhibited (FIG. 23).

Scofaron  contain Production of black mugwort extract

5-1: Preparation of black mugwort extract

(4 kg) purchased from Yeongcheon, Gyeongbuk, was finely divided and 32 L of a first extraction solvent containing 50% alcohol (v / v) was added thereto at a ratio of 1: For 6 hours, and the primary extract was concentrated under reduced pressure to recover.

Next, 20 liters of a secondary extraction solvent containing 50% alcohol (v / v) so as to be a ratio of 1: 5 was added to the sample, and the mixture was extracted at 50 ± 2 ° C. for 16 hours. The secondary extract was concentrated under reduced pressure and recovered . The first concentrated liquid and the second concentrated liquid were mixed and then freeze-dried or spray-dried to prepare a squalona-containing black mugwort extract powder (FIG. 24).

Extraction and concentration of sea squirt Item / Lot Test Example 1 Test Example 2 Test Example 3 Test Example 4 Test Example 5 Remarks Raw material 4kg 4kg 4kg 4kg 4kg Drying and cutting products Primary extraction solvent 32L 32L 32L 32L 32L 50% alcohol (v / v) Primary extraction temperature (℃) 58-62 58-62 58-62 58-62 58-62 1st extraction time 6hr 6 6 6 6 Secondary extraction solvent 20L 20L 20L 20L 20L The first concentrate recovery alcohol Second extraction time 16hr 16hr 16hr 16hr 16hr After 2 hours at 50 ℃ Amount of concentrate (kg) 1.21 1.15 1.24 1.25 1.30 Brix 45 46 46 46 45 moisture(%) 55.95 54.13 54.20 55.80 54.60 Solid content (%) 44.05 45.87 45.80 44.20 45.37 Anhydrous yield (%) 13.32 13.18 14.20 13.81 14.70 Average 13.8%

As shown in Table 2, the extracts of black sea mugwort were extracted by using the extraction solvent containing 50% alcohol in the same manner as above, and the concentration of extract concentrate was measured by lyophilizing each extract. As a result, The average yield was 13.8%.

5-2: contained in the black mugwort extract Scofaron  Content analysis

Each sample was dissolved in 70% MeOH (Table 2), filtered through a 0.45-μm filter membrane, and then filtered through a membrane filter To a concentration of 10 mg / ml and used for HPLC analysis.

A control sample was prepared by finely grinding (1.0 g) a sample of brown iron purchased from Uljin, Gyeongsangbuk-do, and 100 ml of 70% MeOH was treated with ultrasonic method and filtered with a 0.45 μm filter membrane. Finally, the concentration was adjusted to 10 mg / ml and 10 占 퐇 of the filtrate was taken for HPLC analysis.

The content of scopolone contained in the black mugwort extract was analyzed by the HPLC conditions in Table 3.

According to the extraction method of the present invention, the content of scopolone sample Scoparone concentration
(n = 3, 占 퐂 / ml) average con.
(占 퐂 / ml) RSD
(%) content
(Mg / g) Control group 12.1593 12.1362 0.23 1.2136 ± 0.0028 12.1444 12.1049 Test Example 1 62.6324 61.8824 1.31 6.1882 0.0811 61.9928 61.0221 Test Example 2 59.5085 59.7107 0.40 5.9711 + 0.0237 59.6517 59.9719 Test Example 3 53.8737 54.4300 0.91 5.4430 ± 0.0498 54.5837 54.8326 Test Example 4 62.5010 61.9421 1.36 6.1942 0.0841 62.3501 60.9752 Test Example 5 68.4954 67.8892 1.70 6.7889 + 0.1151 68.6101 66.5621

As a result of analysis of the content of scopolone contained in the black mugwort extract, the control group contained about 0.12% by weight of scoparone, while the five mugwort extracts obtained by the method of Example 5-1 contained an average of 0.54 to 0.67% Of scopolone was contained.

Animal experiments Eating efficiency  Affection Scofaron  Effect of the included black mugwort extract

6-1: Breeding and diet of experimental animals

8-week-old female Sprague-Dawley (SD) rats, ovariectomized and sham operated, were purchased from a central laboratory animal and fed with a solid diet (Samyang feed) for 14 days After adjustment, they were divided into 5 groups according to body weight (n = 12 per group).

A total of 84 days of calcium-deficient diets were fed to the experimental animals. For the positive control, 50 μg / kg of 17β-estradiol (E ₂ ) and 125 mg / kg and 250 mg / kg of camphor extract were orally administered daily for 12 weeks Respectively.

Seaweed extract treatment group group process Group I Sham (fake surgery) Group Ⅱ OVA (ovariectomy) Group III OVA + E ₂ (50 μg / kg) Group Ⅳ OVA + black mugwort extract-1 (125 mg / kg) Group V OVA + Black mugwort extract-2 (250 mg / kg)

Dietary intake was measured at a fixed time of 9-10 o'clock every day. The incubation time was adjusted to 12 hours from 9:00 am to 9:00 pm by artificial lighting. The room temperature was maintained at 25 ± 3 ℃ and the relative humidity was 50 ± 10%. Water was used in purified drinking water, feed and water were not restricted, and they were supplied at a fixed time between 9 am and 10 am daily.

6-2: Weight of experimental animals and Eating efficiency  Effect of Fusarium wilt extract

In order to examine the effects of the black mugwort extract containing scopolone on body weight and dietary efficiency of the experimental animals, the body weight of the experimental animals of Example 6-1 was measured, and the average dietary intake during 12 weeks was I (OVA + ACE 50 mg / kg), Group Ⅳ (OVA + ACE 125 mg / kg), Group V (OVA + ACE 250 mg / kg) and Group Ⅱ (OVA) There was no significant difference between the two groups.

However, weight gain was significantly different between group Ⅱ (OVA) and group Ⅳ (Ova + ACE 125 ㎎ / ㎏). It was confirmed that the fat accumulation rate was increased due to ovariectomy and weight gain was observed, and that the black tea extract had no toxicity, no rejection of intake, and no influence on nutritional metabolism (FIG. 25).

Using animal experiments Scofaron  Measurement of Bone Growth and Bone Health Activity of the Snail Extracts Contained

7-1: Kidney, liver, uterus and femur bone of experimental animals ( BMC , BMD ) On Scopa Effect of Rhizobium extract

 At the end of the experiment (12 weeks), the animals were sacrificed and kidney, liver and uterus were excised and left and right femoral bones were extracted and stored at -70 ℃ for analysis.

Bone mineral density (BMD) and bone mineral content (BMC) of the thigh bone were measured using PIXImus (GE medical systems, Lunar, USA). The BMD of the femoral bone tissue is expressed in g units per cm ² , and the BMC in g units.

As shown in Table 7, there was no significant difference in kidney and liver weights. Uterine weight of group Ⅲ (OVA + E2 50 μg / kg) was slightly increased compared with group Ⅱ (OVA) (OVA + ACE 125 mg / kg) and group V (OVA + ACE 250 mg / kg) did not show any significant difference (Fig. 26).

The weight, length, and thickness of the femoral bones were significantly different between group I (Sham) and group Ⅱ (OVA). There was a significant difference between groups Ⅱ and Ⅲ, Ⅳ and Ⅴ I did. However, bone mineral density and mineral content of femur bones decreased in group II (OVA) compared to group I (Sham), but decreased in ovariectomized group (OVA + E2 50μg / : OVA + ACE 250 mg / kg) (Fig. 27).

7-2: Blood of experimental animals PICP ( procollagen type  Ⅰ C- terminal peptide ) Content Scofaron  Effect of the extract of black mugwort

PICP in the blood of experimental animals was measured using enzyme-linked immunosorbent assay (E90570Ra) of USCN. After the animals were lavaged, blood was rapidly collected from the heart and centrifuged at 3,000 rpm for 20 minutes to separate plasma from whole blood. Separated plasma was stored at -70 ° C and used for analysis.

100 [mu] l of plasma diluted 10-fold with PBS in each group was dispensed into all wells except the blank wells of rat PICP coated 96-well strip plates. Gt; 37 C < / RTI > for hours. After removing the diluted plasma, 100 detection of detection reagent A was added and reacted at 37 캜 for 1 hour. Detection reagent A was then removed and each well was washed three times with 350 의 of wash buffer . Then, 100 μl of detection reagent B was added to all wells, reacted at 37 ° C. for 30 minutes, and then each well was washed five times with 350 μl of wash buffer. 90 μl of substrate solution was added to all wells and reacted at 37 ° C. for 20 minutes in the absence of light. After stopping the reaction, 50 μl of stop solution was added, and the reaction was terminated by ELISA reader The absorbance was measured at 450 nm and the calibration curve was generated using the rat PICP standard of 0, 1.25, 2.5, 5, 10, 20, 40 and 80 ng / ml.

As a result, the PICP level of group Ⅲ (OVA + E2 50 ㎍ / ㎏) and Ⅴ group (OVA + ACE 250 ㎎ / ㎏) was significantly increased compared with group Ⅱ (OVA) (Fig. 28), and it was confirmed that administration of the squam powder-containing mugwort extract increases the formation of type I collagen (type I collagen), which can contribute to bone formation.

7-3: Blood of experimental animals BALP ( Bone alkaline 포스화제 ) Content Scofaron  Effect of the extract of black mugwort

Blood plasma BALP of the experimental animals was measured using MBS Bone Alkaline Phosphatase (MBS731429), and the plasma isolated in Example 7-2 was used.

100 쨉 l of a 2-fold diluted plasma of PBS and a conjugate solution were added to all wells except for blank wells of a 96-well plate coated with BALP (BALP coated 96-well strip plate) 50 aliquots were mixed and reacted at 37 캜 for 1 hour. After removing the reaction solution, each well was washed 5 times with 350 μl of wash buffer, and 50 μl of a mixture of the substrate A solution and the substrate B solution in a ratio of 1: 1 was added to each well. The reaction was carried out at 37 캜 for about 15 minutes in the blocked state. After stopping the reaction by stopping 50 μl of the stop solution, the absorbance was measured at 450 nm with an ELISA reader. The absorbance was measured using a BALP standard of 0, 0.5, 1, 2.5, 5 and 10 ng / Respectively.

As a result, BALP activity was significantly higher in group Ⅲ (OVA + E2 50 ㎍ / ㎏), group Ⅳ (OVA + ACE 125 ㎎ / ㎏) and group V (OVA + ACE 250 ㎎ / (Fig. 29). BALP is a bone-specific enzyme secreted during osteoblastic differentiation and is involved in bone remodeling. Increased secretion of BALP by skarfone-containing mugwort extract promotes bone resorption and promotes osteoporosis prevention And can contribute to the treatment.

7-4: Serum of experimental animals OC ( osteocalcin ) Content Scofaron  Effect of the extract of black mugwort

Osteocalcin (OC) in the blood of the experimental animals was measured using a rat osteocla- tin kit (BT-490) from Biomedical Technologies Inc., and the plasma isolated in Example 7-2 was used.

All wells except the blank wells of a 96-well osteocalcin coated 96-well plate coated with mouse osteocalcin were diluted 10-fold with waking sample buffer (working sample buffer BT-471R) 25 ㎕ of plasma and 100 오 of osteocalcin antiserum were added and reacted at 37 캜 for 2 hours and 30 minutes. After washing each well 5 times with 300 μl of wash buffer, 100 μl of donkey anti-Goat IgG peroxidase was added to each well, reacted at room temperature for 1 hour, and then washed with 300 μl of washing buffer And each well was washed five times. 100 μl of a 1: 1 mixture of TMB solution BT-497 (TMB solution BT-497) and hydrogen peroxide solution BT-498 (1: 1) was treated in all wells and reacted at room temperature for 30 minutes in a light- After stopping the reaction by stopping the stop solution, the absorbance was measured at 450 nm with an ELISA reader and the rat osteocalcin standard BT-493 at 0, 0.5, 1, 2.5, 5, 10 and 20 ng / To create a black line.

As a result, in ovariectomized group Ⅱ (OVA), increased osteocalcin level was observed in group Ⅳ (OVA + ACE 125 ㎎ / ㎏) and group V (OVA + ACE 250 ㎎ / Kg) (Fig. 30). This is a decrease in the bone turnover caused by ovariectomy by the extraction of skarfone-containing snow pea extract.

7-5: Blood of experimental animals OPG ( osteoprotegerin ) Content Scofaron  contain Effect of black mugwort extract

In order to examine the OPG regulatory effect of the scarfalo-containing sea anilin extract, blood OPG levels in the experimental animals were measured using the mouse OPG kit (MOP00) of the R & D system, and the plasma isolated in Example 7-2 was used.

50 μl of assay diluent RD1-21 was dispensed into all wells except blank wells of a rat OPG-coated 96-well plate (Rat OPG coated), followed by addition of calibrator diluent RD5-3 to 5 Twenty-five microliters of diluted plasma was added and reacted at room temperature for 2 hours. After removing the reaction solution, each well was washed 5 times with 400 μl of wash buffer, and then 100 μl of a substrate solution was added to all wells and reacted at room temperature for 30 minutes in the state of blocking light. After stopping the reaction with 100 μl of stop solution, the absorbance was measured at 450 nm with an ELISA reader and the mouse OPG standard (0, 31.2, 62.5, 125, 250, 500, 1,000 and 2,000 pg / To create a black line.

As a result, the ovariectomized group Ⅱ (OVA) and ovariectomized group (OVA + ACE 250 ㎎ / ㎏), which were positive control group Ⅲ (OVA + E2 50 ㎍ / It was confirmed that the secretion of OPG was increased (Fig. 31).

7-6: Serum of experimental animals RANKL ( receptor activator of NF -KB ligand ) Content Scofaron  Effect

In order to examine the RANKL modulating effect of the scarfolone-containing sea urchin extract, the blood RANKL concentration in the experimental animals was measured using the mouse TRANCE / RANKL / TNFSF11 kit (MTR00) of the R & D system and the plasma isolated in Example 7-2 Respectively.

50 μl of assay diluent RD1W was dispensed into all wells except the blank wells of 96-well strip plates coated with rat TRANCE (blank wells on Rat TRANCE coated 96-well plate), and then diluted with calibrator diluent RD6-12 25 쨉 l of 2-fold diluted plasma was dispensed and reacted at room temperature for 2 hours. After removing the reaction solution, each well was washed 5 times with 400 μl of wash buffer, and 100 μl of conjugate solution was added thereto. After reacting at room temperature for 2 hours, 400 μl of wash buffer And each well was washed five times. Then, 100 μl of substrate solution was added to all wells and allowed to react at room temperature for 30 minutes in the light-blocked state. After stopping the reaction with 100 μl of stop solution, the absorbance was measured at 450 nm with an ELISA reader and the absorbance was measured at 0, 31.2, 62.5, 125, 250, 500, 1,000 and 2,000 pg / To create a black line.

As a result, ovariectomized group Ⅱ (OVA) and ovariectomized group Ⅳ (OVA + ACE 125 ㎎ / ㎏) and Ⅴ group (OVA + E2 50 ㎍ / + ACE 250 mg / kg), RANKL production was significantly reduced (FIG. 32).

7-7: Blood of experimental animals TRAP ( tartrate resistant acid 포스화제 ) Content Scofaron Containing sea tangle  Effect of extract

 In order to examine the TRAP control effect of the scarfalo-containing sea squirt extract, blood TRAP concentration in the experimental animals was measured using an enzyme-linked immunosorbent assay kit (E90902Ra, USCN), and the plasma isolated in Example 7-2 was used.

100 [mu] l of 5-fold diluted plasma was added to all wells except for the blank wells of a 96-well 96-well strip plate coated with TRAP in rats, Gt; 37 C < / RTI > for hours. Then, 100 detection of detection reagent A was added and reacted at 37 캜 for 1 hour. Then, the reaction solution was removed and each well was washed 3 times with 350 의 of wash buffer. Then, 100 μl of detection reagent B was added to all wells, reacted at 37 ° C. for 30 minutes, and then each well was washed five times with 350 μl of wash buffer. 90 μl of substrate solution was added to all wells and reacted at 37 ° C. for 20 minutes in the absence of light. After stopping the reaction, 50 μl of stop solution was added, and the reaction was terminated by ELISA reader The absorbance at 450 nm was measured and the calibration curve was generated using rat TRAP standard of 0, 0.78, 1.56, 3.12, 6.25, 12.5, 25 and 50 ng / ㎖.

As a result, TRAP content was increased in ovariectomized group Ⅱ (OVA), but the positive control group Ⅲ (OVA + E2 50 ㎍ / ㎏) and scopolone containing black mugwort extract group VI (OVA + ACE 125 ㎎ / And group V (OVA + ACE 250 mg / kg) showed a significant decrease in TRAP content compared to group II (FIG. 33). That is, it was confirmed that the extract of Sophora japonica containing scoparone reduces the osteoclast-induced osteoclast-induced TRAP secretion, thereby preventing and treating osteoporosis.

7-8: Blood ICTP in laboratory animals cross - linked  C- terminal Telopeptide of type Ⅰc ollag en) content Scofaron  Effect

 In order to examine the ICTP regulation effect of the scarfalo-containing sea squirt extract, blood ICTP concentration in the test animal was measured using an enzyme-linked immunosorbent assay kit (E90665Ra, USCN), and the plasma isolated in Example 7-2 was used.

500 쨉 l of plasma diluted 10-fold with PBS for each group was dispensed into all but the blank wells of a rat ICTP-coated 96-well strip plate (Rat ICTP coated 96-well strip plate) , And 50 detection of detection reagent A were dispensed and reacted at 37 캜 for 1 hour. The reaction solution was removed and each well was washed three times with 350 μl of wash buffer. Then, 100 μl of detection reagent B was added to all wells, followed by reaction at 37 ° C. for 30 minutes. Each well was washed five times using wash buffer. 90 μl of substrate solution was added to all wells and reacted at 37 ° C. for 20 minutes in the absence of light. After stopping the reaction, 50 μl of stop solution was added, and the reaction was terminated by ELISA reader The absorbance was measured at 450 nm, and a calibration curve was prepared using rat ICTP standard of 0, 123.5, 370.4, 1,111.1, 3,333.3 and 10,000 pg / ml.

As a result, blood ICTP concentration of ovariectomized group Ⅱ (OVA) was significantly increased compared to group I (sham), but the positive control group Ⅲ (OVA + E2 50 ㎍ / ㎏) Group OVA + ACE 250 mg / kg) significantly decreased ICTP levels compared to group II (OVA) (Fig. 33).

Having thus described a particular portion of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby Will be clear. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (14)

A composition for preventing or treating osteoporosis, which comprises, as an active ingredient, scoparone represented by the general formula (1) or the skarfone-containing sea squirt extract.
[Chemical Formula 1]

[3] The composition for preventing or treating osteoporosis according to claim 1, wherein the content of scopolone or the skarfolone-containing hazelnut extract is from 0.001 to 20% by weight.
The composition for preventing or treating osteoporosis according to claim 1, wherein the soporone or the scarfolone-containing sea squirt extract promotes osteoblast activity and inhibits osteoclast differentiation and bone resorption.
[Claim 2] The composition for preventing or treating osteoporosis according to claim 1, wherein the scarfolone-containing hazelnut extract is prepared by the following steps:
(a) finely finely finely dividing a sample of rosewater, adding a first extraction solvent containing 50% alcohol (v / v) and heating it to 55 to 65 캜 for primary extraction;
(b) recovering and concentrating the primary extract, adding a secondary extraction solvent containing 50% (v / v) vat, and heating to 45 to 55 캜 for secondary extraction; And
(c) recovering said secondary extract, followed by concentration and drying to obtain a skarfone containing musk extract.
5. The method of claim 4, wherein in step (a), the first extraction solvent is added so as to be in a ratio of 1: 7 to 1: 9 with the rhizome sample and then extracted for 4 to 8 hours. Or < / RTI >
5. The method of claim 4, wherein in step (b), the second extraction solvent is added so as to be in a ratio of 1: 4 to 1: 6 with the rhizome sample, and then extracted for 4 to 18 hours. Or a pharmaceutically acceptable salt thereof.
[Claim 5] The composition for preventing or treating osteoporosis according to claim 4, wherein the scarlet wolfberry extract obtained in step (c) contains scopolone in an amount of 5.0 to 7.0 mg / g.
A health functional food for preventing or ameliorating osteoporosis, which comprises scoparone represented by the formula (1) or an extract of a black tea mugwort containing the scopolar as an active ingredient.
[Chemical Formula 1]

[Claim 9] The health functional food for preventing or improving osteoporosis according to claim 8, wherein the content of scopolone or the skarfolone-containing hazelnut extract is 0.001 to 20% by weight.
[Claim 9] The health functional food for preventing or ameliorating osteoporosis according to claim 8, wherein the soporone or the soporax containing safflower extract promotes osteoblast activity and inhibits osteoclast differentiation and bone resorption.
9. The health functional food for preventing or ameliorating osteoporosis according to claim 8, characterized in that the skarfolone-containing hazelnut extract is prepared by the following steps:
(a) finely finely finely dividing a sample of rosewater, adding a first extraction solvent containing 50% alcohol (v / v) and heating it to 55 to 65 캜 for primary extraction;
(b) recovering and concentrating the primary extract, adding a secondary extraction solvent containing 50% (v / v) vat, and heating to 45 to 55 캜 for secondary extraction; And
(c) recovering said secondary extract, followed by concentration and drying to obtain a skarfone containing musk extract.
12. The method of claim 11, wherein in step (a), the first extraction solvent is added so as to be in a ratio of 1: 7 to 1: 9 with the rhizome sample, and then extracted for 4 to 8 hours. Or health functional food for improvement.
12. The method according to claim 11, wherein the second extraction solvent in step (b) is added so as to be in a ratio of 1: 4 to 1: 6 with a rhizome sample, and then extracted for 4 to 18 hours. Health functional foods for prevention or improvement.
12. The health functional food for preventing or ameliorating osteoporosis according to claim 11, wherein the mugwort extract obtained in step (c) contains scopolone in an amount of 5.0 to 7.0 mg / g.

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