KR101282728B1 - Compositions for treatment and prevention of bone diseases comprising extract of thalictrum aquilegifolium var. sibiricum regel ＆ tiling - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of bone diseases comprising the extract of the leg of the pheasant as an active ingredient. The composition according to the present invention reduces the formation of osteoclasts, inhibits bone resorption, and may have the effect of preventing and treating bone diseases associated with bone fractures such as osteoporosis.

Description

COMPOSITIONS FOR TREATMENT AND PREVENTION OF BONE DISEASES COMPRISING EXTRACT OF THALICTRUM AQUILEGIFOLIUM VAR. SIBIRICUM REGEL & TILING}

The present invention relates to a composition for the prevention and treatment of bone diseases comprising the extract of the leg of the pheasant 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. Two of the most important 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. Currently, bisphosphonate preparations are administered in high doses, which has resulted in a significant increase in jaw bone necrosis patients. Therefore, it is a very important task to find a substance which is complementary to the disadvantages of bisphosphonates and which is not toxic 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 due to chemicals. On the other hand, Pheasant's leg extract is extracted from natural materials and has the same effect on bone loss as bisphosphonate preparations. .

It is an object of the present invention to provide a substance that is effective for bone resorption by osteoclasts without side effects and toxicity using extracts of natural pheasant legs.

In one embodiment, in order to achieve the above object, it provides a pharmaceutical composition for the prevention and treatment of bone diseases containing the extract of Thalictrum aquilegifolium as an active ingredient. In another embodiment, the bone disease is any one selected from the group consisting of osteoporosis (osteoporosis), osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease and metabolic bone disease. It provides a pharmaceutical composition. In another embodiment, the bone disease provides a pharmaceutical composition for the prevention and treatment of bone diseases, characterized in that caused by metastasis of cancer cells. In another embodiment, the extract provides a pharmaceutical composition for the prevention and treatment of bone diseases, characterized in that the inhibition of the formation of osteoclasts. In another embodiment, the extract provides a pharmaceutical composition for the prevention and treatment of bone diseases, characterized in that inhibiting bone resorption by osteoclasts. In another embodiment, the bone disease provides a pharmaceutical composition for the prevention and treatment of bone diseases, characterized in that osteoporosis (osteoporosis).

In one embodiment, the Pheasant's Leg ( Thalictrum) It provides a food composition for the improvement and alleviation of bone diseases containing aquilegifolium ) extract as an active ingredient. In another embodiment, the bone disease is any one selected from the group consisting of osteoporosis (osteoporosis), osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease and metabolic bone disease, improvement and alleviation It provides a food composition for. In another embodiment, the bone disease provides a food composition for improving and alleviating bone disease, characterized in that caused by bone metastasis of cancer cells. In another embodiment, the extract provides a food composition for the improvement and alleviation of bone diseases, characterized in that the inhibition of the formation of osteoclasts. In another embodiment, the extract provides a food composition for improving and alleviating bone disease, characterized in that the bone resorption by osteoclasts. In another embodiment, the bone disease provides osteoporosis (osteoporosis), and provides a food composition for improving and alleviating bone diseases.

In one embodiment, the present invention provides a method of treating bone diseases by administering an effective amount to the subject.

In one embodiment, the present invention provides a method of inhibiting the formation of osteoclasts by administering to the subject an effective amount of the extract.

In one embodiment, the present invention provides a method of inhibiting bone uptake by osteoclasts by administering an effective amount to the subject.

In the present invention, "Thalictrum aquilegifolium" is a perennial herb and grows in the shade of mountain forests of about 50-100 cm in height. The leaves are regenerated, the entire triangle is divided into two or three feathers, and the leaves at the bottom are long, but shorter as they go up. The chin leaves are membranous and flat, flipped back, and there are chin leaves. Small leaf is obovate, triangular, divided into 4, and rounded. The flower is white and has a conical inflorescence, without petals. Calyx leaves are 4 ~ 5 oval, reddish before blooming. The stamens are many and the upper part of the operating table spreads out to form a spatula, anther is yellow. The achene is obovate or elliptical with 3-4 ridges. In the outpost, 0.1% of the root contains 0.07% of alkaloids such as talomin, talomidine, isocoridine, magnoflorin and flavonoids, quercetin, camphorol and the organic acids caffeic acid, P-coumaric acid, cinafinic acid and ferulic acid. Etc. Folks cut fresh starch juice or root powder, apply inflammatory drugs to wounds, swelling, uterine bleeding, and wound wounds with medicines and blood drops. Outpost rain forest drugs to drink lung diseases, root decoction is used as a pain medicine for gastritis and chest pain, dysmenorrhea. It is also used for diarrhea and vision problems. In addition, it is also used for jeongan, neuropathy, swelling.

In the present invention, "bone disease" is a result of the imbalance between osteoclasts and osteoblasts in bone, but is not limited thereto. Bone damage, osteoporosis, osteomalacia, rickets disease, fibrous osteoarthritis caused by bone metastasis of cancer cells , Aplastic bone disease and metabolic bone disease (see Korean Patent Nos. 10-0399374 and 10-0720026).

The composition according to the present invention reduces the formation of osteoclasts, inhibits bone resorption, and may have the effect of preventing and treating bone diseases associated with bone fractures such as osteoporosis.

1 is a graph showing the cell viability of mouse bone marrow macrophages by the leg extract of the pheasant.
Figure 2 is a result image and graph showing the inhibitory effect of the extract of the pheasant on osteoclast formation.
Figure 3 is an image showing the inhibitory effect of the extract of the pheasant's leg on bone uptake by osteoclasts.

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 Pheasant Leg Extract

Harvested Pheasant's Leg ( Thalictrum) aquilegifolium , Rural Development Administration, Crop Science Institute , Suwon-si , Gyeonggi-do, Korea. Selected, washed and dried in a 50 ℃ drier (DS 80-2; Dasol Science, Korea). ; Daesung Atron, South Korea). 54 g of powder of the leaf of the pheasant and 59 g of the powder of the stem were each accelerated solvent extractor [ASE (Accelerated Solvent Extractor) -300; DIONEX, USA], the extract obtained by repeatedly extracting twice with 50% 100% methanol at 50 ℃ for 5 minutes using a vacuum condenser (SB-1000; EYELA, Japan) while maintaining the concentration below 50 ℃ to extract finally 7.61 g and 1.64 g were obtained respectively.

Example  2: Mouse Bone Marrow Macrophage Culture

The cervical spine of three-week-old male ICR mice (Central Experimental Animal Co., Ltd., Korea) was dislocated 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. 500 μl of α-MEM was placed in a 1 ml syringe, and a syringe needle was inserted into the spinal cord in 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 a medium containing Histopaque (Sigma, USA). The isolated bone marrow macrophages were treated with 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: Cytotoxicity Measurement of Pheasant Leg Extract in Mouse Bone Marrow Macrophages

MTT assay was performed to determine the cytotoxicity of the pheasant's leg extract against mouse bone marrow macrophages. Pheasant leg extract was dissolved in DMSO (dimethyl sulfoxide; Sigma, USA) and then 5, 10 and 20 μg, respectively, with α-MEM added with 1% antibiotic-antibacterial solution, 10% FBS and M-CSF (30 ng / ml). Dilute to a concentration of / ml. After 5 × 10 ⁴ mouse bone marrow macrophages were put into each well of a 96-well plate, 200 μl of α-MEM containing the diluted extract was added thereto to culture the mouse bone marrow macrophages. Every two days it was exchanged with fresh α-MEM medium containing pheasant leg extract. After 5 days of incubation in a cell incubator at 37 ° C. and 5% CO ₂ , MTT (3- (4,5-dimethythiazol-2-yl) -2,5-diphenyl tetrazolium bromide was added at a concentration of 5 mg / ml for each well. 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 20 minutes the absorbance was measured at 570 nm. Cell viability was calculated as the percentage of absorbance of the experimental group (well treated extract) relative to the absorbance of the control group (well treated only medium).

The cytotoxicity of the pheasant's leg stem extract against mouse bone marrow macrophages did not decrease the cell viability of bone marrow macrophages when treated at concentrations of 5, 10 and 20 μg / ml. That is, the pheasant extracts did not show cytotoxicity against myeloid macrophages (FIG. 1 and Table 1).

Pheasant's Leg Stem Extract Concentration (μg / ml) Absorbance (570nm) Cell survival rate of mouse bone marrow macrophages (%) 0 0.656 100 5 0.698 106.5 10 0.718 109.4 20 0.662 100.9

Example  4: Mouse Osteoclast Formation of Pheasant Leg Extract Inhibition  Measure

In order to confirm the inhibitory effect of pheasant's leg stem extract 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). Pheasant leg stem extract was dissolved in DMSO and then 5, 10, 20 μg / ml with α-MEM added with 1% antibiotic-antibacterial solution, 10% FBS, RANKL (100ng / ml) and M-CSF (30ng / ml). Dilute to a concentration of. The isolated bone marrow macrophages were cultured by adding 5 × 10 ⁴ cells to each well in a 96-well plate and adding 200 μl of α-MEM depending on the diluted concentration. As a control, only the α-MEM medium solution containing no pheasant leg extract was added. The 96-well plate was replaced with the same α-MEM medium containing the extract every two days, and then cultured in a cell incubator at 37 ° C., 5% CO ₂ for 5 days, followed by a TRAP assay kit (tartarate resistant) to confirm osteoclast formation. Multinucleated osteoclasts were stained using an 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 bone marrow macrophages that were not treated with RANKL, whereas osteoclasts were significantly increased in bone marrow macrophages treated with RANKL. On the other hand, bone marrow macrophages treated with RANKL and 5, 10, and 20 μg / ml of pheasant leg stem extract significantly reduced osteoclast formation compared to bone marrow macrophages treated with RNAKL alone. The formation of osteoclasts decreased by 14.04% at 5 μg / ml, 28.42% at 10 μg / ml, and 52.79% at 20 μg / ml. In conclusion, the stem extracts of pheasant significantly reduced osteoclast formation in a concentration-dependent manner (Fig. 2 and Table 2).

Pheasant's Leg Stem Extract Concentration (μg / ml) Mouse osteoclast count Mouse osteoclast
Formation inhibition rate (%) 0 477 ± 17 - 5 410 ± 18 14.04 10 342 ± 31 28.42 20 225 ± 11 52.79

Example  5: Bone Resorption of Osteoclasts by Leg Stem Extract of Pheasant Inhibition

In order to confirm whether the pheasant's leg stem extract showed an 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, 5 × 10 ⁴ macrophages (BMMs) isolated from the 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 two days it was replaced with fresh medium and incubated for 4 days in a cell incubator at 37 ℃, 5% CO ₂ conditions. After confirming the formation of osteoclasts, a medium containing pheasant leg stem extract was added. Pheasant leg stem extract was dissolved in DMSO and added to 1% antibiotic-antibacterial solution, 10% FBS, RANKL (100ng / ml), and M-CSF (30ng / ml). Dilute to Again, every two days, the fresh medium containing the extract was exchanged and bone uptake by osteoclasts was measured until day 15-17. 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, uptake and production increased compared to the group not containing RANKL. However, when the RANKL and pheasant leg stem extracts were treated as compared to the RANKL-treated control group, it was confirmed that the uptake was suppressed. Therefore, the leg extract of the pheasant not only inhibited the formation of osteoclasts but also inhibited bone resorption by the osteoclasts (FIG. 3).

Example  6: 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. After two oral administrations of the extract of Example 1 at a dose of 1 g / kg to two animals in each group, the mortality, clinical symptoms, and weight changes of the animals were observed, and hematological and blood biochemical tests were performed. The necropsy was performed to visually check the tonic and thoracic organ abnormalities. 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.

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 (12)

Osteoporosis, osteomalacia, rickets disease, fibrous osteoarthritis, aplastic bone disease and metabolic bone disease, which contain the extract of Thalictrum aquilegifolium as an active ingredient Pharmaceutical composition. delete The method of claim 1,
The bone disease is a pharmaceutical composition for the prevention and treatment of bone disease, characterized in that caused by the metastasis of cancer cells. The method of claim 1,
The extract is a pharmaceutical composition for the prevention and treatment of bone diseases, characterized in that to inhibit the formation of osteoclasts. The method of claim 1,
The extract is a pharmaceutical composition for the prevention and treatment of bone diseases, characterized in that to suppress bone resorption by osteoclasts. The method of claim 1,
Wherein the bone disease is osteoporosis. ≪ RTI ID = 0.0 > 25. < / RTI >
Osteoporosis, osteomalacia, rickets disease, fibrous osteoarthritis, aplastic bone disease and metabolic bone disease, which contain the extract of Thalictrum aquilegifolium as an active ingredient Food composition. delete 8. The method of claim 7,
The bone disease is a food composition for improving and alleviating bone diseases, characterized in that caused by bone metastasis of cancer cells. 8. The method of claim 7,
The extract is a food composition for improving and alleviating bone diseases, characterized in that to inhibit the formation of osteoclasts. 8. The method of claim 7,
The extract is a food composition for the improvement and alleviation of bone diseases, characterized in that to suppress bone resorption by osteoclasts. 8. The method of claim 7,
Food composition for the improvement and alleviation of bone diseases, characterized in that the bone disease is osteoporosis (osteoporosis).

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