KR101320975B1 - Composition for treatment and prevention of bone diseases comprising extract of magnoliae flos's active components - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of bone disease, wherein the composition contains the active compound as an active ingredient, the composition of the present invention is less toxic, effective bone disease by inhibiting the formation of osteoclasts and bone resorption by osteoclasts Therapeutic agents may be provided. In addition, it is possible to supplement problems such as jaw bone necrosis and incapacitation of bones or joints, which are disadvantages of bisphosphonate-based therapeutic agents used in recent years for treating bone damage.

Description

COMPOSITION FOR TREATMENT AND PREVENTION OF BONE DISEASES COMPRISING EXTRACT OF MAGNOLIAE FLOS'S ACTIVE COMPONENTS}

The present invention relates to a composition for preventing and treating bone diseases, wherein the god contains the active compounds as an active ingredient.

The reduction of bone density and bone mass, a symptom of osteoporosis, is a result of the imbalance of osteoclasts and osteoblasts in the bone. Excessive osteoclast production leads to diseases that reduce bone density, such as osteoporosis. Osteoclasts that perform bone resorption are large, multinucleated cells that break down bone matrices and break down bone minerals. Activated osteoclasts have three or more nuclei, which require various hormones and factors to induce differentiation from osteoclast precursor cells to mature multinucleated osteoclasts. The two most important of these factors are macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL) produced from osteoblasts (Mojtaba A. et al., Cancer Biol) Ther., 7: 1,3-9; 1 (2008)).

M-CSF is a cytokine expressed from osteoblasts and stromal cells and plays an important role in osteoclast formation (Anne T. et al., J Surg Res., 100: 18-24 (2001)). . M-CSF primarily plays an important role in cell proliferation, survival and cytoskeletal organization (Kim SY. Et al., J Korean Orthop Assoc., 44: 151-158 (2009)). Another important factor, RANKL, is expressed on the surface of osteoblasts and is released by activated T-cells. RANKL attaches to RANK receptors in osteoclast progenitor cells to induce and differentiate osteoclast growth (Mojtaba A. Cancer biology & Therapy 7: 1,3-9; 1 (2008)). RANKL promotes the differentiation of osteoclasts by activating transcription factors such as c-fos, nuclear factor of activated T cells (NFATc1) and Nuclear factor kappa B (NF-kB), phosphatidylinositol 3-kinase (PI-3K), It activates signaling systems such as extracellular signal-regulated kinase (ERK) to promote osteoclast survival and function (LEE ZH. Et al., Biochem Biophys Res Commun., 305: 211-213 (2003)).

As such, bone uptake by enzymes secreted from osteoclasts induces osteoporosis and pain and fracture, and osteoclasts have been designated as target cells for treating them. In other words, if the osteoclast differentiation is inhibited, bone resorption can be suppressed and bone diseases and osteoporosis can also be treated. Currently, bisphosphonate-based therapeutic agents such as Fosamax (aldronate) and Actonel (ingredient name: risedronate) are widely used for the treatment of osteoporosis such as osteoporosis by osteoclast. Most of these bisphosphonate agents act to slow down or stop bone loss by weakening the functions of osteoclasts that destroy bone and inducing apoptosis. However, these drugs do not have the ability to promote new bone formation, and there are an increasing number of cases of osteonecrosis, severe atrial fibrillation, incapacitation of bones or joints, or musculoskeletal pain in patients who have recently taken bisphosphonates. (Coleman RE., Br J Cancer, 98: 1736-1740 (2008)). In particular, osteoclast formation is promoted by cancer cells that have metastasized to bone in breast cancer and prostate cancer patients, and serious bone diseases occur. There is no drug for treating them. High doses of bisphosphonate preparations are currently being used, which has led to a marked increase in patients with jaw bone necrosis (Smith MR. Cancer Treat Rev 31 (suppl 3): 19-25 (2005); Lipton A, Cancer 88: 1082-1090 (2000). Berenson JR, et al. Cancer 91: 1191-1200 (2000); Abrahamsen B. Calcif Tissue Int .; 86: 421-435 (2010)). Therefore, it is a very important task to find a substance that is effective in supplementing the disadvantages of bisphosphonates, nontoxic and effective for bone resorption by osteoclasts.

The solution to this problem is to use extracts extracted from natural substances. Plant-derived substances are less toxic than most chemically-synthesized drugs, and they are also in the spotlight because they can prevent genetic modification in the body. On the other hand, the active compounds isolated from the gods are extracted from natural substances, have the same effect on bone loss as the bisphosphonate preparations, and are not toxic and can be used to supplement the problems seen in bisphosphonate preparations such as jaw bone necrosis and bone neutralization. It is expected.

It is an object of the present invention to provide an effective composition for bone diseases such as osteoporosis using active compounds isolated from a natural extract, Sinyi.

In order to achieve the above object, in one embodiment of the present invention provides a pharmaceutical composition for the prevention and treatment of bone diseases containing the following formula (1) as the active ingredient is an active compound.

(1)

However, in Formula 1, R ₁ to R ₃ are OCH ₃ or H. In another embodiment, R ₁ and R ₃ of Formula 1 are OCH ₃ Or H and R ₂ is OCH ₃ to provide a pharmaceutical composition for preventing and treating bone disease. In another embodiment, the god active compound provides a pharmaceutical composition for preventing and treating bone diseases, characterized in that the god is isolated from the extract. In another embodiment, the bone disease is selected from the group consisting of bone damage caused by bone metastasis of cancer cells, osteoporosis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease and metabolic bone disease It provides a pharmaceutical composition for the prevention and treatment of bone diseases. In another embodiment, the bone disease provides a pharmaceutical composition for the prevention and treatment of bone diseases, characterized in that osteoporosis. In another embodiment, the god active compound provides a pharmaceutical composition, characterized in that it inhibits the formation of osteoclasts. In another embodiment, the god active compound provides a pharmaceutical composition, characterized in that it inhibits bone resorption by osteoclasts.

In one embodiment, the present invention provides a health functional food for preventing and alleviating bone diseases, which contains the following Chemical Formula 1 as an active ingredient:

(1)

Provided that R ₁ to R ₃ are OCH ₃ or H. In another embodiment, R ₁ and R ₃ of Formula 1 are OCH ₃ or H, R ₂ OCH ₃ provides a dietary supplement for the prevention and alleviation of bone diseases. In another embodiment, the god active compound provides a dietary supplement for the prevention and alleviation of bone diseases, characterized in that the god is isolated from the extract. In another embodiment, the bone disease is selected from the group consisting of bone damage caused by bone metastasis of cancer cells, osteoporosis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease and metabolic bone disease Provided is a dietary supplement for the prevention and alleviation of bone diseases. In another embodiment, the bone disease provides a dietary supplement for the prevention and alleviation of bone diseases, characterized in that osteoporosis. In another embodiment, the god active compound provides a dietary supplement for the prevention and alleviation of bone diseases, characterized in that the inhibition of the formation of osteoclasts. In another embodiment, the god active compound provides a dietary supplement for the prevention and alleviation of bone diseases, characterized in that the bone resorption by osteoclasts.

Magnoliae Flos of the present invention is Magnolia denudata DER.) or other buds of the same genus (Magnolia and Magnoliaceae). Magnolia is a deciduous tree of the genus Magnoliaceae. The nature is warm and nonpoisonous, the taste is spicy. Entering menopause and gastric diameter in 12 menstrual veins to show the efficacy of gongpungtonggyu (祛風 通竅), sore head pain, bleeding (rhinitis, rhinitis), coloration (鼻塞, nasal congestion), leuko turbidity, toothache, etc. To cure. Sinyi is known as an excellent herbal medicine to treat rhinitis.

The term " bone disease " in the present invention is a result of imbalance between osteoclasts and osteoblasts in bone, including but not limited to bone damage caused by cancer cells osteoporosis, osteoporosis, osteomalacia, rickets, , Intractable bone disease, and metabolic bone disease (see Korean Patent Nos. 10-0399374 and 10-0720026).

The composition of the present invention, the total active weight of the composition is 0.1 to 50% by weight. The composition comprising the god active compound of the present invention may further comprise a suitable carrier, excipient or diluent according to conventional methods. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The compositions according to the invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories or sterile injectable solutions, respectively, according to conventional methods. have. More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, and the like in the active compound. It can be prepared by mixing sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The present invention may be varied depending on the age, sex and body weight of the patient, but it is generally administered in an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, once to several times per day . The dosage may also be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition 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 or intracerebroventricular injections. Since the active compound of the present invention has little toxicity and side effects, it can be used with confidence even for long-term use for prophylactic purposes.

The present invention provides a dietary supplement containing the active compound and food supplements, which are food acceptable, and various foods, for example, beverages, gums, teas, vitamin complexes, dietary supplements, etc. It may be used in the form of a powder, granule, acupuncture, tablet, capsule or beverage. At this time, the amount of the god active compound in the food or beverage can be generally added to 0.01 to 15% by weight of the total food weight in the case of the health food composition of the present invention, 0.02 to 10 based on 100 ml for the health beverage composition g, preferably 0.3 to 1 g.

Food supplementary additives as defined herein include food additives customary in the art, such as flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like. The health beverage composition according to the present invention is an essential ingredient in the ratio indicated, and there is no particular limitation on the ingredients to which the god is added in addition to the active compound, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, like ordinary drinks. . Examples of the natural carbohydrate include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrins; And sugar alcohols such as xylitol, sorbitol, and erythritol. As natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The composition of the present invention containing the active compounds as active ingredients is less toxic and can provide an effective bone disease treatment agent by inhibiting the formation of osteoclasts and bone resorption by osteoclasts.

In addition, it is possible to supplement problems such as jaw bone necrosis and incapacitation of bones or joints, which are disadvantages of bisphosphonate-based therapeutic agents used in recent years for treating bone damage.

1 is a graph showing the cell viability of mouse bone marrow macrophages according to the amount of the active compound added to the kidney.
Figure 2 is a photograph showing the inhibitory effect of the active compound god on osteoclast formation.
Figure 3 is a graph showing the inhibitory effect of God active compound on osteoclast formation.
Figure 4 is a photograph showing the inhibitory effect of the god active compound on bone uptake by osteoclasts.
5 is a diagram showing the effect of the god active compound on the activity of MMP secreted from osteoclasts.
Figure 6 is a graph showing the effect of the god active compound on the activity of Cathepsin K secreted from osteoclasts.
7 is a graph showing the effect of kidney extract in ovarian extraction induced osteoporosis animal model.
8 is a graph showing the cell survival rate of human breast cancer cells by the renal active compound.
Figure 9 is a diagram showing the effect of the renal active compounds on the mRNA expression of osteolytic factors secreted from breast cancer cells.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

Example  1. Preparation of God Active Compounds

The Manchunhua ( Magnolia biondii , 10.45 kg) purchased from the market was extracted three times with 60 L of MeOH for 5 hours at 60 ° C., and the extract was concentrated using a rotary vacuum evaporator to obtain 864.91 g of MeOH extract.

Network pornographic the MeOH extract in the same way and then hexane layer was dissolved in water 10ℓ one ml of hexane 10ℓ herein fraction Hexane and an aqueous layer in fraction funnel was concentrated under reduced pressure to give the hexane fractions 156.6 g again the water layer and the CH ₂ Cl ₂ 10 ℓ, EtOAc 10 ℓ, n-BuOH 10 ℓ fractionated CH ₂ Cl ₂ 458.93 g of fractions, 26.4 g of EtOAc fraction, 59.44 g of n-BuOH fraction and 153 g of water fraction were obtained. 65 g of CH ₂ Cl ₂ fractions were separated by silica gel column. Fill the column (length 80cm, diameter 10cm) with 25 cm of silica gel (0.063-0.200 mm) and the solvent starts to flow out of 100% hexane mobile phase to increase the polarity in the order of hexane: EtOAc = 1: 1 and then CH ₂ Cl _The distillation was started again with ₂ 100%, and CH ₂ Cl ₂ : MeOH = 7: 1 in the order of increasing polarity to obtain a small fraction 1-18.

Of these, small fraction 5 was recrystallized from MeOH to obtain fargesin.

Subfraction 8 was extracted with solvent hexane: CHCl ₃ : EtOAc = 15: 10: 1 using silica gel (0.063 mm or less) column chromatography to obtain a total of four subfractions, the fourth of which was dissolved in MeOH. After adsorbing completely on RP-18 column which was flowed out with H ₂ O 100%, it was flowed with 60% H ₂ O to obtain aschantin.

Subfraction 13 was extracted with solvent hexane: CHCl ₃ : EtOAc = 10: 10: 1 using silica gel (0.063 mm or less) column chromatography to obtain a total of seven subfractions. The sixth subfraction was recrystallized from MeOH. Lirioresinol B dimethyl ether was obtained.

Subfraction 15 was separated by silica gel (0.063 mm or less) column chromatography using solvent hexane: CHCl ₃ : EtOAc = 30: 10: 1 to obtain a total of seven subfractions, the fifth of which was dissolved in MeOH. recrystallization from a small fraction was discharged to 60% H ₂ O was completely adsorbed to the sephadex LH-20 column which flows out to the H ₂ O 100% then with MeOH to give the magnolin.

The structure of each compound was confirmed by comparing the mass spectrometry and NMR data of each compound with those reported in the literature (Li J et al., Chem. Pharm. Bull. 53: 235-237 (2005); Youn U et al. 56: 115-117 (2008); Schuehly W et al., Nat. Prod. Commun. 4: 231-234 (2009); Lee J et al., Chem. Pharm. Bull. 57: 298-301 (2009).

Example  2. Mouse Bone Marrow Macrophage Culture

Three-week-old male ICR mice (Central Experimental Animal Co., Ltd., Korea) were dislocated from the cervical spine and the outer leg of the hind leg was removed using a forceps. The exfoliated hind legs were cut with surgical scissors and placed in serum-free α-MEM (Minimum Essential medium alpha; Gibco, USA). Using tweezers, the bone in the muscle was separated and transferred to fresh α-MEM. 600 μl of α-MEM was placed in a 1 ml syringe, and a syringe needle was inserted into the spinal cord of the center of the leg bone and sprayed 2-3 times to extract bone marrow cells. Collected bone marrow cells were collected through a centrifuge and mixed with fresh α-MEM. Bone marrow macrophages were then isolated from the extracted bone marrow cells using separation medium Histoque (Sistmaque, USA). The isolated myeloid macrophages were 1% Antibiotic-Antimycotic (Gibco, USA), 10% fetal bovine serum (FBS); Gibco, USA), macrophage-colony stimulating factor (M-CSF); R & D system Inc, USA) was incubated in α-MEM containing 30ng / ml.

Example  3. Measurement of Cytotoxicity of Renal Active Compounds in Mouse Bone Marrow Macrophages

MTT assay was performed to determine the cytotoxicity of renal active compounds on mouse bone marrow macrophages. God dissolved the active compounds in DMSO (dimethyl sulfoxide; Sigma, USA), followed by α-MEM with 1% antibiotic-antibacterial solution, 10% FBS and M-CSF (30 ng / ml), respectively. Dilute to concentration. After inserting 1 × 10 ⁴ mouse bone marrow macrophages into each well of a 96-well plate, 200 μl of α-MEM containing diluted active compounds were added thereto to culture the mouse bone marrow macrophages. Every two days, gods were exchanged with fresh α-MEM medium containing active compound, 10% FBS and M-CSF (30 ng / ml). After incubation for 5 days in a cell incubator at 37 ° C., 5% CO ₂ , MTT (3- (4,5-dimethythiazol-2-yl) -2,5-diphenyl tetrazolium to a concentration of 0.45 mg / ml per well bromide (Sigma, USA) solution was added. After incubation at 37 ° C. for 4 hours, the medium and MTT solution were completely removed and 200 μl of DMSO was added per well. After 30 minutes the absorbance was measured at 570 nm. Cell viability was calculated as the percentage of absorbance of the experimental group (well God treated active compounds) relative to the absorbance of the control group (well treated only medium).

The cytotoxicity of renal active compounds against mouse bone marrow macrophages did not significantly reduce cell viability of bone marrow macrophages when treated at concentrations of 1, 10 and 20 μM. That is, the active compounds did not show cytotoxicity against myeloid macrophages (FIG. 1 and Table 1).

God active compound concentration
(μM) Absorbance (570nm) Cell survival rate of mouse bone marrow macrophages (%) Control group 0 0.893 100.0 Aschatin One 0.837 93.6 10 0.836 93.6 20 0.950 106.7 Fargesin One 0.957 107.1 10 0.873 97.7 20 0.819 91.7 Lirioresinol B dimethyl ether One 0.826 92.4 10 0.831 93.0 20 0.905 101.3 Magnolin One 0.868 97.2 10 0.948 106.2 20 0.908 101.6

Example  4. Mouse osteoclast formation of renal active compounds Inhibition  Measure

In order to confirm the inhibitory efficacy of the renal active compounds on RANKL-induced osteoclast formation in mouse bone marrow macrophages, experiments were performed as described in the literature (Park EK.et.al., Biochem Biophys Res). Commun., 325 (4): 1472-1480 (2004). After the active compounds were dissolved in DMSO, α-MEM added 1% antibiotic-antibacterial solution, 10% FBS, RANKL (100ng / ml) and M-CSF (30ng / ml). Dilute to a concentration of μM. The isolated bone marrow macrophages were placed in 96-well plates and 1 × 10 ⁴ cells per well were incubated with 200 μl of α-MEM depending on the diluted concentration. The control group added only α-MEM media solution (1% antibiotic-antibacterial solution, 10% FBS and M-CSF (30ng / ml) but no RANKL) containing no active compounds. 96-well plates were exchanged every 2 days with the same α-MEM medium containing 1% antibiotic-antibacterial solution, 10% FBS, RANKL (100 ng / ml) and M-CSF (30 ng / ml) and material, 37 After culturing for 5 days in a cell incubator at 5 ° C. and 5% CO ₂ condition, multinucleated osteoclasts were stained using a TRAP assay kit (tartarate resistant acid phosphatase assay kit; Sigma, USA). Three or more osteoclasts in the nucleus were counted using an optical microscope.

As a result, osteoclasts were not produced in the bone marrow macrophages (control group) not treated with RANKL, but the number of osteoclasts was significantly increased in the bone marrow macrophages treated with RANKL (p <0.0001). On the other hand, in bone marrow macrophages treated with RANKL with active compounds 1, 5 and 10 μM, osteoclast formation was significantly reduced in a dose dependent manner compared to bone marrow macrophages treated with RNAKL alone (FIGS. 2, 3 and Table 2).

God active compound concentration
(μM) Mouse osteoclast count Mouse osteoclast formation inhibition rate (%) Control group 0 - - RANKL 0 378 ± 3 0 RANKL + Aschatin One 345 ± 8 8.6 5 236 ± 3 37.6 10 139 ± 1 63.1 RANKL + Fargesin One 362 ± 2 4.1 5 154 ± 7 59.2 10 87 ± 3 77.0 RANKL + Lirioresinol B dimethyl ether One 305 ± 3 19.2 5 283 ± 5 25.1 10 162 ± 2 57.1 RANKL + Magnolin One 392 ± 5 0 5 325 ± 7 13.9 10 271 ± 4 28.3

Example  5. Bone resorption of osteoclasts by renal active compounds Inhibition

In order to confirm whether the active compounds exhibited inhibitory effect on bone resorption by RANKL-induced osteoclasts, the following experiment was conducted according to the method described in the literature (Myung Hee Kim, J Cell Physiol., 221: 618-628. (2009)). In a 16-well plate (Biocoat osteologic multitest slides; BD, USA) specially coated with calcium phosphate, 1 × 10 ⁴ macrophages (BMM) isolated from bone marrow of three-week-old mice were placed in each well. 200 μl of α-MEM added with 1% antibiotic-antibacterial solution, 10% FBS, RANKL (100ng / ml) and M-CSF (30ng / ml) was added. On the other hand, only the α-MEM medium containing no RANKL was added to the control group. Every 2 days, exchange with fresh medium containing 1% antibiotic-antibacterial solution, 10% FBS, RANKL (100ng / ml) and M-CSF (30ng / ml) and 4 days in a cell incubator at 37 ° C and 5% CO ₂ Incubated. After confirming the formation of osteoclasts, a medium containing renal active compounds was added. The active compounds were dissolved in DMSO and α-MEM added with 1% antibiotic-antibacterial solution, 10% FBS, RANKL (100ng / ml) and M-CSF (30ng / ml). Dilute to concentration. It was also replaced with fresh medium containing extract every two days and incubated until day 15. After 15 days the medium was removed and added with sodium hypochlorite solution. After 5 minutes, the sodium hypochlorite solution was completely removed, washed twice with distilled water, and the absorption pit formed by the osteoclasts was observed under an optical microscope.

As a result, when RANKL was treated to bone marrow macrophages, the production of absorption pores was increased compared to the group not containing RANKL. However, compared to the RANKL-only group, it was confirmed that the production of absorption holes was suppressed when RANKL and Shin were treated with the active compounds together. Therefore, the active compounds not only inhibited the formation of osteoclasts but also inhibited bone resorption by osteoclasts (FIG. 4).

Example  6. God is secreted from osteoclasts by active compounds MMP -9 and MMP Activity inhibitory activity

Gelatin zymography was performed to confirm the inhibitory effect of the active compounds on the expression of MMP-9 and MMP-2, which are released from osteoclasts and affect bone resorption. The active compounds were dissolved in DMSO (dimethyl sulfoxide (Sigma, USA)) and added α-MEM with 1% antibiotic-antibacterial solution, 10% FBS and M-CSF (30 ng / ml). Dilute to a concentration of. The isolated bone marrow macrophages were incubated in 96-well plates with 5 × 10 ⁴ cells per well and 200 μl of α-MEM depending on the diluted concentration. As a control, only the α-MEM medium solution containing no active compounds was added. The 96-well plate was exchanged with the same α-MEM medium containing the extract every two days, and cultured for 5 days in a cell incubator at 37 ° C. and 5% CO ₂ conditions to confirm osteoclast formation. Cultures in 96-well plates were obtained and centrifuged using a centrifuge to remove impurities, followed by supernatant. 10% sodium dodecyl sulfate (SDS) -polyacrylamide gel containing 5% gelatin was prepared, and the protein in the culture was quantified by BSA protein assay, and the amount of protein was corrected to 35 μl. After mixing the corrected culture and sample dye, 20 μl of the prepared mixture was loaded and electrophoresed at 60V. After washing the gel with wash buffer, incubation buffer was added and incubated for 24 hours in a constant temperature water bath at 37 ℃, 50rpm conditions. After 24 hours, the gel was washed, stained for 1 hour using a commassie blue solution, and decolorized twice for 30 minutes using a decolorizing solution to observe a change in MMP expression in the gel.

Investigating the effects of active compounds on the expression of MMP-2 and MMP-9 secreted by osteoclasts and related to bone resorption by osteoclasts, MMP- was found in the RANKL-treated group compared to the non-RANKL-treated group. 9 activity was significantly increased. But MMP-2 did not change much. In the group treated with active compounds, the expression level of MMP-9 was significantly reduced compared to the group treated with RANKL. In contrast, MMP-2 did not decrease (FIG. 5). In other words, the active compound of the god had an effect on MMP-9, which is known to play an important role in osteoclast capacity of osteoclasts, but did not affect MMP-2.

Example  7. God is secreted from osteoclasts by active compounds Cathepsin  K active Inhibition  Measure

To determine the inhibitory effects of God-activated compounds on Cathepsin K activity, which is released from osteoclasts and affects bone resorption, 1% antibiotic-antibacterial solution, 10% after dissolving the active compounds in DMSO (dimethyl sulfoxide; The active compounds were diluted to 5 and 10 μM with α-MEM with FBS and M-CSF (30 ng / ml). The isolated bone marrow macrophages were incubated in 96-well plates with 5 × 10 ⁴ cells per well and 200 μl of α-MEM depending on the diluted concentration. The control group added only α-MEM medium solution containing no active compounds. The 96-well plate was exchanged every 2 days with the same α-MEM medium containing the active compounds, and after 6 days incubation in a cell incubator at 37 ℃, 5% CO ₂ conditions confirmed the formation of osteoclasts. On day 6, cultures in 96-well plates were obtained, and the supernatants were obtained after centrifugation using a centrifuge to remove impurities. Changes in Cathepsin K activity were measured in osteoclast supernatants recovered using a Sensizyme Cathepsin K activity assay kit (Sigma-Aldrich, USA). The recovered osteoclast supernatant and Cathepsin K standard were placed in a 96-well plate containing Cathepsin K antibody and reacted at room temperature for 1 hour, then removed and washed with washing buffer, and then the reaction mixture was added at 37 ° C for 2-4 hours. Reacted. Cathepsin K activity was calculated by reading the absorbance at 405 nm and substituting the Cathepsin K standard curve.

Experimental results showed that Cathepsin K activity was decreased in a concentration-dependent manner in the group treated with active compounds compared with RANKL-only group. The graph shows the value corrected through protein quantification of the activity of Cathepsin K (Table 3 and Figure 6).

God active compound
Concentration (μM) Cathepsin K Activity
(pg / mg protein) Cathepsin K inhibition rate (%) Control group 0 0.2 - RANKL 0 5.3 - RANKL + Aschatin 10 2.9 45 20 1.6 70 RANKL + Fargesin 10 3.3 37 20 1.8 66 RANKL + Lirioresinol B dimethyl ether 10 2.7 48 20 1.1 80 RANKL + Magnolin 10 1.6 70 20 0.9 83

Example  8. Against Osteoporosis Induced by Ovarian Extraction Sini extract Inhibition

In 8-week-old female ICR mice isolated from ovaries (Central Laboratory Animal, Inc., Korea), estrogen secretion is inhibited, leading to osteoporosis. Therefore, we investigated whether osteoporosis is inhibited by oral administration of kidney extract in ovarian isolated mouse osteoporosis model. Ten female ICR 8-week-old mice were assigned to each group and divided into groups as follows.

Control: No surgical treatment.

Sham: Opened, but not ovarian.

OVX: Ovarian extraction and feeding vehicle 5 days a week.

OVX + E2: Ovarian extraction and subcutaneous injection of 17β-estradiol at 10 μg / kg body weight (b.w) 5 days a week.

OVX + Renal Extract 0.1 mg / kg b.w: Ovariant extraction and oral administration of Renal Extract of specified concentration 5 days a week.

OVX + Renal Extract 1 mg / kg bw: Ovarian extraction followed by oral administration of Renal Extract at the indicated concentration 5 days a week.

After the oral administration of sinyi extract for 60 days, the weight of the mice were weighed, blood was drawn from the heart, centrifuged at 3000 rpm and 4 ° C for 20 minutes, and serum was separated and stored at -80 ° C. Bone production and bone lysis indices, alkaline phosphatase (ALP), calcium, tartrate-resistant acid phosphatase form 5b (TRAP5b), and osteocalcin were measured using a kit.

ALP was used QuantiChrom Alkaline Phosphatase Assay Kit (BioAssay System, USA). 200 μl of water, calibrator and 5 μl of sample were placed in a 96-well plate and working solution was added to the well containing the sample to 200 μl. Absorbance was measured at 405 nm (t = o) and again 4 minutes later (t = 4). The ALP activity was determined by substituting the values given in the data sheet of the kit.

Calcium was used QuantiChrom Calcium Assay Kit (BioAssay System, USA). 5 μl of standard and sample were added to a 96-well plate, 200 μl of working solution was added, incubated for 3 minutes, and the absorbance was measured at 612 nm. Calculated the calcium concentration of the sample by drawing a standard curve and substituting the equation.

TRAP5b was used with a MouseTRAP Assay kit (IDS Ltd, USA). 100 μl of Anti-MouseTRAP antibody was placed on an Antibody coated plate and incubated with shaking at room temperature for 60 minutes. After rinsing the plate with the wash buffer, 100 μl of Calibrator and Control were put in the plate, and 75 μl of 0.9% NaCl solution was added to the other well and 25 μl of sample was placed on top. 25 μl of Releasing reagent was added to all wells and incubated with shaking at room temperature for 60 minutes. After rinsing the plate with a washing buffer, 100 μl of Substrate solution was added and incubated at 37 ° C. for 2 hours. 25 μl of Stop solution was added and the absorbance was measured at 405 nm. Using the Calibrator, the formula was calculated and substituted to determine the activity of TRAP5b.

Osteocalcin was used in the Mouse Osteocalcin EIA kit (Biomedical Technologies Inc., USA). 25 μl of sample buffer (Blank), Standard, Cintrol, and samples were placed in a 96-well plate provided in the kit and 100 μl of osteocalcin antiserum was placed thereon. The lid was touched and incubated at 4 ° C. for 24 hours. After rinsing the plate using a washing buffer, 100 μl of Streptavidin-Horseradish Peroxidase was added and incubated at room temperature for 30 minutes. TMB solution and Hydrogen Peroxide solution were mixed at a ratio of 1 to 1, and the plates were rinsed, and 100 μl of the prepared solution was added and reacted at room temperature and in a dark place (aluminum foil) for 15 minutes. Then 100 μl of stop solution was added and the absorbance was measured at 450 nm. Using the standard curve, the equation was calculated and the sample value was substituted to determine the activity of osteocalcin.

The results showed that OVX increased body weight and significantly increased serum calcium levels, ALP activity, osteocalcin, and TRAP5b activity in ovary extraction group. Increasing indices of osteoporosis were significantly suppressed. Sinyi extract showed an osteoporosis inhibitory effect similar to the effect observed in the estrogen-administered group (FIG. 7).

Example  9. Measurement of Cytotoxicity of God-Active Compounds in Human Breast Cancer Cells

MTT assay was performed to confirm the cytotoxicity of renal active compounds against human breast cancer cells (MDA-MB-231). Sincin dissolves the active compounds in DMSO (dimethyl sulfoxide; Sigma, USA), and then adds 1% antibiotic-antibacterial solution and 10% FBS to DMEM medium, respectively, which shows 10, 20, 40, 60, 80 and 100 μM Dilute to concentration. Each well of a 96-well plate was placed in 200 μl medium (FBS10%) in 1 × 10 ⁴ MDA-MB-231 cells for 24 h, followed by 200 μl of DMEM medium (serum free) containing the diluted active compounds. Incubated in a cell incubator at 37 ° C., 5% CO ₂ conditions for 24 h and 72 h, followed by MTT (3- (4,5-dimethythiazol-2-yl) -2, to a concentration of 0.45 mg / ml per well. 5-diphenyl tetrazolium bromide (Sigma, USA) solution was added. After incubation at 37 ° C. for 4 hours, the medium and MTT solution were completely removed and 200 μl of DMSO was added per well. After 30 minutes the absorbance was measured at 570 nm.

The treatment of cytotoxicity with MDA-MB-231, a human breast cancer cell, for 24 hours and 72 hours, and the results showed that the active compounds did not significantly reduce the cell viability of breast cancer cells (Fig. 8 and Table 4). .

time God active
compound God active compound concentration
(μM) Absorbance (570nm) Cell survival rate of breast cancer cells (%) 24h Aschatin 0 0.395 100 10 0.398 100.7 20 0.38 96 40 0.406 102.6 60 0.398 100.6 80 0.426 107.7 100 0.369 93.3 Fargesin 0 0.602 100 10 0.693 115.2 20 0.671 111.6 40 0.671 111.5 60 0.643 106.8 80 0.55 91.4 100 0.548 91.1 Lirioresinol B dimethyl ether 0 0.386 100 10 0.396 101.7 20 0.382 99 40 0.373 96.5 60 0.358 92.6 80 0.341 88.2 100 0.338 87.5 Magnolin 0 0.359 100 10 0.357 99.3 20 0.35 97.4 40 0.35 97.4 60 0.347 96.7 80 0.295 82.1 100 0.286 79.7 72h Aschatin 0 0.397 100 10 0.512 128.9 20 0.505 127.1 40 0.492 124 60 0.467 117.5 80 0.43 108.4 100 0.356 89.8 Fargesin 0 1.008 100 10 0.996 98.8 20 0.962 95.5 40 0.817 81.1 60 0.844 83.8 80 0.773 76.7 100 0.513 51 Lirioresinol B dimethyl ether 0 0.412 100 10 0.484 117.6 20 0.491 119.1 40 0.474 115.1 60 0.395 95.9 80 0.309 75 100 0.299 72.6 Magnolin
0 0.425 1000 10 0.484 114.1 20 0.484 114.1 40 0.452 106.6 60 0.426 100.4 80 0.397 93.5 100 0.331 77.9

Example  10. Bone soluble factors secreted from human breast cancer cells mRNA  God for expression of active compounds Inhibition

Osteolytic factors secreted by cancer cells increase RANKL in osteoblasts, and it is known that RANKL binds to RANK of osteoclasts and promotes osteoclast formation. Since there is no therapeutic agent for bone metastasis of cancer cells, the clinic uses bisphosphonate, which is a treatment for osteoporosis, to treat it, and it is known that the incidence of jaw bone necrosis is significantly higher than that of osteoporosis patients because it is used at a high dose in cancer patients. Since the extract of Sinyi or the active ingredient of Sinyi inhibits osteoclast formation as well as the expression of osteolytic factors in cancer cells, it is thought that it is more effective in treating bone damage caused by metastasis of cancer cells in cancer patients. Current bisphosphonate formulations reduce pain in cancer patients and are considered to be ineffective for life extension. In other words, the following experiments were conducted to show that cancer cells are effective in inhibiting bone damage caused by metastasis to bone.

To examine the mRNA expression of osteolytic factor, reverse transcriptase-polymerase chain reaction (RT-PCR) was performed. MDA-MB-231 cells (1x10 ⁶ cells) were placed in a 75T flask and cultured in L-15 medium containing 10% FBS. When the cells grew about 80-90%, the culture medium was removed and God was incubated for 24 hours in L-15 (0% FBS) containing the active ingredients. Breast cancer cells were collected and total RNA was isolated using TRIZOL reagent (Life Technologies, Grand Island, NY, USA). CDNA was synthesized by performing reverse transcriptase reaction on the reaction mixture (20 μl) containing the isolated RNA, oligo-dT15, ribonuclease inhibitor, and reverse transcriptase. cDNA was amplified in a PCR reaction mixture containing cDNA, primers (10 pmol), dNTP, MgCl 2, Tag DNA polymerase. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as a control, and the base sequences of the primers used were as follows (Table 5).

mRNA Primer sequences Annealing Temp. Expected Size (bp) PTHrP sence: 5'-AACTCGCCTCCAACCTGCGC-3 ' 62 ℃ 212 antisence: 5'-CGCTCGGGACCTCCTCTGGT-3 ' IL-1β sence: 5'-ATGGCAGAAGTACCTAAGCTC-3 ' 56 ℃ 810 antisense: 5'-TTAGGAAGACACAAATTGCATGGTGAACTCAGT-3 ' IL-6 sence: 5'-CATCCTCGACGGCATCTCAGC-3 ' 60 ° C 332 antisense: 5'-TTGGGTCAGGGGTGGTTATTG-3 ' IL-8 sence: 5'-ATGACTTCCAAGCTGGCCGT-3 ' 56 ℃ 285 antisence: 5'-CCTCTTCAAAAACTTCTCCACACC-3 ' IL-11 sence: 5'-AGCCACCACCGTCCTTCCAAA-3 ' 60 ° C 351 antisense: 5'-CCTCCGTCCCCACCCCAACAT-3 ' GAPDH sence: 5'-CCGCCTACTGCCCACTGCCACCAC-3 ' 52 ℃ 420 antisense: 5'-TCCATCCACTATGTCAGCAGGTCC-3 '

PCR products were confirmed by using a UV transilluminator after electrophoresis on hardened 2% agarose gel with ethidium bromide. The intensity of the bands was measured using image analysis software (TINA, version 2.0). Intensity of osteolysis factor bands was normalized compared to GAPDH band intensities.

A study was performed on RT-PCR at a concentration that did not affect cell viability at 24 hours, and the effects of god active components on mRNA expression of bone soluble factor mRNA in MDA-MB-231 breast cancer cells were examined. In IL-1β, the expression of PTHrP mRNA was significantly reduced (FIG. 9).

Example  11: Bone disease  In vivo administration of extracts for treatment

The acute toxicity test was carried out as described below using specific pathogen-free (SPF) SD rats of 6 weeks old supplied from the experimental supply center. Two animals in each group were orally administered with the renal active ingredient of Example 1 at a dose of 1 g / kg, and then examined for mortality, clinical symptoms, and weight changes of the animals. Necropsy was performed to visually observe abnormalities in the organs and thoracic organs. As a result of the experiment, there were no clinical symptoms or dead animals in all animals treated with the test substance, and no toxic change was observed in weight change, blood test, blood biochemical test, and autopsy findings. As a result, it was confirmed that the extract of the present invention did not exhibit any toxicity at 1 g / kg or more in rats, and that the oral LDL was at least 1 g / kg.

Examples of formulations for the composition of the present invention are illustrated below. However, the following formulation examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following formulation examples.

Formulation example  One: Sanje  Produce

God active compound 300 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2: Preparation of tablets

50 mg of active compounds

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3: Preparation of capsules

50 mg of active compounds

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4: Preparation of injection

50 mg of active compounds

Appropriate sterile distilled water for injection

pH adjuster

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation example  5: Liquid  Produce

God active compound 100 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6: Manufacture of health food

God Active Compound 1000mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

10 μg biotin

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium Pantothenate 0.5mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7: Manufacture of health drinks

God Active Compound 1000mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to a conventional healthy beverage manufacturing method, and then stirred and heated at 85 ° C. for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 l container, sealed sterilized and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

While the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. You will know. In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention be construed as including all embodiments falling within the scope of the appended claims.

Claims (14)

Bone damage, osteoporosis, osteomalacia, rickets disease, fibrous osteoarthritis, amorphous bone disease and metabolic bone disease caused by bone metastasis of cancer cells containing God as an active compound Pharmaceutical composition for the prevention and treatment of bone diseases, which is selected from the group consisting of.
(1)

Provided that R ₁ to R ₃ are OCH ₃ or H. The method of claim 1,
R ₁ and R ₃ of Formula 1 OCH ₃ Or H, R ₂ is OCH ₃ pharmaceutical composition for the prevention and treatment of bone diseases. The method of claim 1,
The god active compound is a pharmaceutical composition for preventing and treating bone diseases, characterized in that the god is isolated from the extract. delete The method of claim 1,
The bone disease is a osteoporosis pharmaceutical composition for the prevention and treatment of bone diseases, characterized in that. The method of claim 1,
The god active compound is a pharmaceutical composition, characterized in that to inhibit the formation of osteoclasts. The method of claim 1,
The renal active compound is a pharmaceutical composition, characterized in that to inhibit bone resorption by osteoclasts. Bone damage, osteoporosis, osteomalacia, rickets disease, fibrous osteoarthritis, amorphous bone disease and metabolic bone disease caused by bone metastasis of cancer cells containing God as an active compound Health functional food for bone disease prevention and alleviation will be selected from the group consisting of.
(1)

Provided that R ₁ to R ₃ are OCH ₃ or H. The method of claim 8,
R ₁ and R ₃ of Formula 1 OCH ₃ Or H, R ₂ is OCH ₃ health functional food for the prevention and alleviation of bone diseases. The method of claim 8,
The god active compound is a health functional food for the prevention and alleviation of bone diseases, characterized in that the god is isolated from the extract. delete The method of claim 8,
The bone disease is a health functional food for the prevention and alleviation of bone diseases, characterized in that osteoporosis. The method of claim 8,
The god active compound is a health functional food for the prevention and alleviation of bone diseases, characterized in that to inhibit the formation of osteoclasts. The method of claim 8,
The renal active compound is a health functional food for the prevention and alleviation of bone diseases, characterized in that the bone resorption by osteoclasts.

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