KR101056362B1 - A composition for the prevention or treatment of bone disease, using Platicodin D as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of bone diseases comprising Platicodin D, one of the saponin components of Giltyeong, as an active ingredient. The composition of the present invention exhibits very good efficacy in inhibiting osteoclast formation and reducing bone uptake by osteoclasts and consequently inhibiting bone destruction. The present invention provides basic data as medicines and foods of Platicodin D having the efficacy of preventing and treating bone diseases caused by bone diseases such as osteoporosis and bone metastasis of cancer cells.

Platicodin D, Gilt, Bellflower, Osteoclasts, Bone Resorption, Osteoporosis, Bone Disease

Description

Composition or composition for preventing or treating bone disease using Platicodin D as an active ingredient {Compositions for Prevention or Treatment of Bone Diseases Comprising Platycodin D}

The present invention relates to a composition for the prevention and treatment of bone diseases comprising Platicodin D as an active ingredient.

Bones support the soft tissues and weight of the body and surround internal organs to protect internal organs from external shocks. It is also an important part of the human body that not only structurally supports muscles or organs but also stores calcium and other essential minerals in the body, such as phosphorus and magnesium. Thus, the bones of grown adults do not stop, and until the day they die, they maintain a very dynamic and continuous regeneration of the process of producing and absorbing the old bones and replacing them with new bones. This is called bone remodeling (Yamaguchi A. et al., Tanpakushitsu Kakusan Koso. , 50 (6Suppl): 664-669 (2005)). Bone turnover, which removes old bone and replaces it with new bone, is essential for restoring bone damage caused by growth and stress and maintaining proper bone function (Cohen-Solal M. et al., Therapie . , 58: 391-393 (2003).

It is known that two types of cells are involved in aggregate formation. One of the two cells is an osteoblast that produces bone, and the other is an osteoblast that destroys bone. Osteoblasts produce RANKL (receptor activator of nuclear factor-κB ligand) and osteoprotegerin (OPG), its decoy receptor. When RANKL binds to RANK, a receptor on the surface of osteoclast progenitor cells, osteoclast precursor cells mature into osteoclasts, resulting in bone resorption. However, when OPG binds to RANKL, the binding between RANKL and RANK is blocked, which suppresses the formation of osteoclasts and prevents bone resorption from occurring more than necessary (Theill LE. Et al., Annu Rev Immunol. , 20: 795-823 (2002). Wagner EF. Et al., Curr Opin Genet Dev., 11: 527-532 (2001)). Absorption or destruction of old bone is caused by osteoclasts from blood cells (hematopoietic stem cells), which are used to puncture bone and release small amounts of calcium into the bloodstream to maintain body function (William J. et al. , Nature., 423: 337342 (2003). Osteoblasts produced from bone cells, on the other hand, fill the pores with collagen and cover the calcium and phosphorus (hydroxyapatite) to form new bones that are hard to rebuild the skeleton (Stains JP. Et al., Birth Defects Res C Embryo Today. , 75 ( 1): 72-80 (2005)). It takes about 100 days for bones to begin to break down and remodel into new bones (Schwarz EM. Et al., Curr Opin Orthop ., 11: 329-335 (2000)). In infants, bone calcium changes 100% within a year, but in adults, about 10-30% of the skeleton is remodeled through this process every year. Many important diseases can be caused when the important bones are out of balance. In particular, diseases related to bone damage due to osteoporosis and bone metastasis of cancer cells are typical.

Osteoporosis is a condition in which bone mass decreases due to various causes and the risk of fracture is continuously increased due to the deterioration of the microstructure of bone tissue. Osteoporosis is a condition in which minerals (particularly calcium) and substrates that make up bone are reduced. Formation is broken and osteoclasts occur in an increased state than osteoblasts (Iqbal MM., South Med J. , 93 (1): 2-18 (2000)). Normal bone inside has a dense structure like a net, but in the case of osteoporosis, the gap between the structures becomes wider and the microstructure becomes thinner and weaker, so that the bone can be easily fractured even with a small impact (Stepan JJ. Et al. , Endocr Regul. , 37 (4): 225-238 (2003) Osteoporosis develops rapid bone loss (2-3% per year) at the onset of menopause and increases the risk of vertebral compression and carpal fractures. Postmenopausal osteoporosis, which occurs slowly in men and women over 70 years of age (0.5-1% per year), and in older age of osteogenic osteoporosis resulting in progressive bone loss of the hip and vertebrae, Regardless of age, diseases (endocrine diseases, gastrointestinal diseases, malignancies) or drugs (adrenal cortex hormones, chemotherapy, thyroid hormones, anticonvulsants, anticoagulants, methotrexate, Claus tarpaulins (cyclosporine), GnRH, etc.), alcohol, smoking, and (is classified as Secondary osteoporosis) (Rosen CJ, N Engl J Med, 353 (6..) 2 primary osteoporosis caused by accidents: 595-603 (2005 Davidson M., Clinicain Reviews. , 12 (4): 75-82 (2002)).

Bone metastases almost always occur in patients with breast cancer, prostate cancer or multiple myeloma (Kozlow W. et al., J Mammary Gland Biol Neoplasia. , 10 (2): 169- 180 (2005)), how long these cancer patients can live is known to be affected by bone metastasis. The reason why the mortality rate increases in breast cancer patients and prostate cancer patients is because cancer cells selectively metastasize to bone. Most bone metastases observed in breast cancer are osteolytic metastasis that destroy bones and are known to occur when breast cancer cells stimulate osteoclasts rather than directly affect bone (Boyde A. et al., Scan Electron Microsc . , 4: 1537-1554 (1986)). On the other hand, bone metastases observed in prostate cancer are osteoblastic metastasis. Osteoblastic bone metastasis is also known to be closely associated with osteolysis. Cancer cells that have metastasized to bone proliferate in the microenvironment around the bone and stimulate the activity of osteoclasts or osteoblasts to determine whether they progress to osteolytic bone metastasis or osteoblast metastasis (Choong PF. Et al., Clin Orthop). Relat Res. , 415S: S19-S31 (2003)). About 80% of breast cancer patients develop bone metastasis of cancer cells, and the metastasized breast cancer cells activate osteoclasts (Bendre M., et al., Clin Orthop Relat Res., 415 (Suppl): S39-S45 (2003) Palmqvist P. et al., J Immunol. , 169 (6): 3353-3362 (2002)). Activated osteoclasts disrupt the balance of the microenvironment around bones, leading to osteolysis, which often causes pathological fractures, as well as leukoerythroblastic anaemia, bone malformations, and hypercalcemia. ), Bone-related diseases such as pain, neuro-compression syndromes (Roodman GD., N Engl J Med. , 350: 1655-1664 (2004)).

Currently, bisphosphonate-based therapies such as Fosamax (component name: alendronate) and Actonel (component name: risedronate) are used for bone damage caused by osteoporosis and bone metastasis of cancer cells. Most of these bisphosphonate preparations work to slow or stop bone loss by weakening and inducing the death of osteoclasts that destroy bone. Recently, however, there have been an increasing number of cases of osteonecrosis, severe atrial fibrillation, incapacitation of bones or joints, or musculoskeletal pain in patients taking bisphosphonates (Coleman RE., Br J Cancer , 98: 1736-1740). (2008) Therefore, much attention has been paid to the development and prevention of osteoporosis prevention and treatment that can effectively suppress bone resorption with fewer side effects.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors made an intensive study to develop a substance that is safe for the human body, especially a plant-derived substance, which can effectively treat or prevent a bone disease, and as a result, one of the effective ingredients of Gilkyung, which is conventionally used as a herbal medicine or folk medicine, By discovering that ticodine D is very good at treating or preventing bone diseases, the present invention has been completed.

Accordingly, an object of the present invention is to provide a composition for preventing and treating bone disease, which comprises Platicodin D as an active ingredient.

Another object of the present invention to provide a method for preventing or treating bone disease.

Another object of the present invention is to provide a novel use for the manufacture of a drug (medicament) for the prevention or treatment of bone diseases.

Still another object of the present invention is to provide a food composition for preventing and treating bone disease, which contains Platicodin D as an active ingredient.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a composition for preventing or treating bone disease comprising Platicodin D as an active ingredient.

According to another aspect of the present invention, the present invention provides a method for preventing or treating bone disease, comprising administering to a subject a composition comprising Platicodin D as an active ingredient.

According to another aspect of the invention, the invention provides the use of Platicodin D for the manufacture of a medicament for the prevention or treatment of bone diseases.

The present inventors have tried to develop a substance that is safe for human body and can effectively treat or prevent bone diseases, especially plant-derived substances. As a result, Platycodin D, which is an effective ingredient of Gilkyeong, which has been conventionally used as a herbal medicine or folk medicine, It has been found to be very effective in treating or preventing a disease.

Platicodine D used in the present invention is one of the active ingredients included in the Platycodon grandiflorum (Platycodi Radix). Gilkyung is a perennial herb that belongs to the bellflower family, which grows and is planted in mountains of various regions. The roots of Gil-gil contain about 2% of saponins. When the water is decomposed, polygalaic acid C ₃₀ H ₄₈ O ₆ and bellflower saponenin C ₃₀ H ₄₈ O ₇ , platyogeninic acid A, B, C, platicodige Become Nin. In addition, it contains phytosterols, small amounts of alkaloids, large amounts of inulin, and platicinine (C ₆ H ₁₀ O ₅ ) ₁₀ . Platicodine D is a major component of triterpene saponins and is known to be effective in lowering anti-inflammatory, anti-allergic and cholesterol levels. Recently, it has been reported to induce the death of human leukemia cells. (Hai Lin Zhao, et al., European Journal of Phamacology , 537: 166-173 (2006); Kwang Seok Ahn, et al., Life Sciences, 76: 2315-2328 (2005); Mun-Ock Kim, et al. , Int. J. Cancer, 122: 2674-2681 (2008).

As used herein, the term 'gilpyeong extract' means that it is obtained by extracting from various organs or parts (eg leaves, flowers, stems, etc.) of the Giltyeong, preferably the extract obtained from the ground or root, most preferably root it means.

The composition of the present invention contains a Gilyeong extract as an active ingredient. As used herein, the term 'extract' is used to refer to Gilgyeong, as well as the extraction result obtained by treating the extraction solvent in Gilgyeong as well as means that it is processed (eg, powdered) Gilgyeong processed to be administered to the animal itself. Has

In the case of obtaining the Gil-gil extract used in the composition of the present invention by treating the Gil-gil extract with various solvents, various extraction solvents may be used. Preferably, a polar solvent or a nonpolar solvent can be used. Suitable polar solvents include (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol Or ethylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO) and (v) dimethyl sulfoxide (DMSO). Suitable as nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- Pentene, 1-chlorobutane, 1-chloropentane, o -xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloro Methane, 1,2-dichloroethane, anneal, diethylamine, ether, carbon tetrachloride and THF.

More preferably, the extraction solvent used in the present invention is (a) water, (b) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) the lower alcohol and water Mixed solvent with (d) acetone, (e) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) hexane and (j) diethyl ether Include. Most preferably, the extract of the present invention is obtained by treating water, butanol, ethyl acetate, or a combination thereof over the street.

As used herein, the term "extract" has the meaning commonly used as a crude extract in the art as described above, but also broadly includes a fraction additionally fractionating the extract. That is, the Giltyeong extract includes not only one obtained by using the above-described extraction solvent, but also one obtained by additionally applying a purification process thereto. For example, fractions obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. The fraction obtained through the purification method is also included in the Giltung extract of the present invention.

The gilyeong extract used in the present invention may be prepared in powder form by an additional process such as distillation under reduced pressure and freeze drying or spray drying.

The composition for preventing or treating bone diseases of the present invention treats or prevents various bone diseases through the action of reducing osteoclasts induced by RANKL (receptor activator of NF- [kappa] B ligand).

According to a preferred embodiment of the present invention, examples of bone diseases that can be prevented or treated by the present invention include bone damage caused by bone metastasis of cancer cells, osteoporosis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease, Metabolic bone disease, osteolysis, leukopenia, malformations of bone, hypercalcemia or neurocompression syndrome.

In particular, the composition of the present invention shows an excellent effect in the prevention and treatment of osteoporosis.

Osteoporosis can be divided into primary (primary) osteoporosis and primary (secondary) osteoporosis. Primary osteoporosis refers to osteoporosis, which can occur mainly in women and older men after menopause, inadequate intake of calcium and vitamin D, inadequate physical exercise, smoking, or smoking. Secondary osteoporosis means that osteoporosis is caused by certain diseases or drugs. In this case, osteoporosis can be induced in young people, and the bone strength decreases and the incidence of fracture increases in proportion to the severity of the disease or drug and the duration of exposure. Diseases include hyperthyroidism, parathyroidism, Cushing syndrome (adrenal cortical hormone secretion), early menopause, menopause caused by artificial surgery (ovarian resection), hypogonadism, chronic liver disease (cirrhosis), rheumatoid arthritis, chronic renal failure , Gastrectomy, etc., and drugs include steroids (corticosteroids), anticonvulsants (epilepsy drugs), heparin, and the like.

The composition of the present invention shows an excellent effect in the prevention and treatment of bone diseases caused by bone metastasis of cancer cells or cancer tissues. For example, the cancer cells or cancer tissue may be gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, rectal cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, cervical cancer, endometrial cancer, brain cancer, prostate cancer, bone cancer , But not limited to, skin cancer, thyroid cancer, leukemia, non-Hodgkin lymphadenopathy, parathyroid cancer, and ureter cancer cells or tissues.

According to an embodiment of the present invention, the composition of the present invention exhibits the effect of reducing the formation of osteoclasts and inhibiting bone resorption and bone destruction by osteoclasts.

Osteoclasts are cells produced from blood cells (hematopoietic stem cells), and have a function of destroying bones when calcium lacks in blood or when bone remodeling is required. In addition, the term "bone uptake" as used herein means that osteoclasts break down bone by secreting strong acid and protease into bone.

The composition of the present invention may be prepared as a pharmaceutical composition.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of Platicodine D of the present invention as described above; And (b) a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of Platicodin D described above.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and preferably applied by oral administration.

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.001-100 mg / kg on an adult basis.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

The composition of the present invention may be provided as a food composition.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a food-effective amount of the above-described placodin D of the present invention; And (b) a food acceptable carrier.

When the composition of the present invention is prepared as a food composition, it contains not only Platicodin D as an active ingredient, but also components commonly added in food production, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings and Contains flavors. Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared with a drink, it further includes citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, tofu extract, jujube extract, licorice extract, etc. You can.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides a composition for the prevention and treatment of bone diseases using Platicodin D.

(Ii) The composition of the present invention exhibits great efficacy in inhibiting bone resorption by reducing the formation of osteoclasts.

(Iii) The present invention provides basic data as medicines and foods of Platicodin D having efficacy for preventing and treating bone diseases caused by osteoporosis and bone metastasis of cancer cells.

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 embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1 Preparation of Platycodin D

1-1. Isolation of Platicodin D

Platicodin D is a "gilsaponin-high fraction containing fractions using high-speed countercurrent chromatography from the tillage extract and a method for separating and producing a high purity platicodine D from the fraction in large quantities" (Korean Patent Application No. 10-2008-0081605 Platicodine D was selectively isolated from the placoside-containing fraction using high-speed counter chromatography according to Example 4 of (C). Or Analytical Science Vol. 3, No. 3, pp. 399-404 (1990), Korean Society of Analytical Science; Or Dong-Yoon Lim et al., Korean J. Physiol. Pharmacol. , Platicodin D was selectively isolated according to the method disclosed in 7: 149-155 (2003). Or Analytical Science Vol. 3, No. 3, pp. 399-404 (1990), Korean Society of Analytical Science; Or Dong-Yoon Lim et al., Korean J. Physiol. Pharmacol. , Can be selectively separated according to the method disclosed in 7: 149-155 (2003).

Ethyl acetate: butanol: water (1: 1: 2, v / v) solvent system with an appropriate K-value was used, and the separation of high-speed countercurrent chromatography was performed by using the upper layer of the solvent as the fixed phase and the lower layer as the mobile phase. After filling the coil with fixed phase and mobile phase ratio of 70:30, equilibrating between the stationary phase and the mobile phase in the column, inject 250 mg of gilsaponin dissolved in 20 ml of the mobile phase, and set the mobile phase speed to 1.3 ml / min for 360 minutes. Shed for a while. After confirming that the eluate of the high-speed countercurrent chromatography was detected by an evaporative light scattering detector ELSD, only 25 mg of pure Platicodine D was obtained at a retention time of 250-280 minutes.

1-2. Purity Measurement of Platicodin D

HPLC-ELSD was used to measure the purity of the concentrated Platicodine D, and (A) water and (B) acetonitrile were mixed by gradient in the mobile phase. This gradient method uses (A) water and (B) acetonitrile for 0-6 minutes (10-15% B), 6-50 minutes (15-25% B), and 50-60 minutes (25-47.5% B). ). Nitrogen was used as the nebulizer gas, flow rate was 1 ml / min, ELSD system was 70 °, gain was 6, and the pressure was adjusted to 2.2 bar. It was.

As a result, it was confirmed that Platicodin D showed a high purity of 95% in the purity measurement experiment of Platicodin D from long saponin using high-speed countercurrent chromatography.

Example 2: Mouse Bone Marrow Macrophage Culture

Four-week-old male (male) ICR mice (Central Experimental Animal Co., Ltd., Korea) were removed from the hind limb by using the forceps after the cervical spine. The exfoliated hind limb was cut with surgical scissors and placed in serum-free α-MEM (Minimum Essential Medium Alpha; Gibco, USA). Tweezers were used to separate the bones in the muscle and transfer them to fresh α-MEM. 500 μl of α-MEM was put into a 1 ml syringe, and a syringe needle was inserted into the spinal cord of the center of the leg bone and sprayed twice to extract bone marrow cells. Bone marrow macrophages were isolated from the extracted bone marrow cells using a separation medium histopark (Histopaque; Sigma, USA). Isolated mouse 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 Systems Inc., USA, were cultured in α-MEM containing 30 ng / ml.

Example 3: Culture of MDA-MB-231 Cells, Human Breast Cancer Cell Line

Human breast cancer cells, MDA-MB-231 cells (1 × 10 ⁶ cells; KCLB, Korea), were treated with 1% antibiotic-antibacterial (100 units / ml penicillin, 100 μg / ml streptomycin) in a 100 pi cell culture dish. Dulbecco's Modified Eagle Medium (DMEM) containing 0.25 μg / ml amphotericin B) solution and 10% FBS; Gibco, USA) cultures.

Example 4 Measurement of Cytotoxicity of Platycodin D in Mouse Bone Marrow Macrophages

In order to confirm the cytotoxicity of Platicodin D on mouse bone marrow macrophages, experiments were performed as described in the literature (Wattel A. et al., J Cell Biochem. , 92 (2): 285-) . 295 (2004). Platicodine D was dissolved in DMSO (dimethyl sulfoxide; Sigma, USA), and then α-MEM with 1% antibiotic-antibiotic solution, 10% FBS and M-CSF (30 ng / ml) was added to 0, 0.1, 0.5, Dilutions were made at concentrations of 1, 3, 5 and 10 μM. Mouse x-ray bone marrow macrophages were cultured by adding 1 × 10 ⁴ mouse bone marrow macrophages into each well of a 96-well plate, and then adding 200 μl of α-MEM each containing diluted placodin D. The 96-well plates were replaced with fresh α-MEM medium added with 1% antibiotic-antibiotic solution, 10% FBS and M-CSF (30 ng / ml) every other day, and the cell incubator at 37 ° C. and 5% CO ₂ conditions. After 5 days of incubation, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide; Sigma, USA) solution was added to a concentration of 5 mg / ml for each well. 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 with media containing Platicodin D) relative to the absorbance of the control group (well treated only medium).

The cytotoxicity of Platicodin D against mouse bone marrow macrophages was shown to increase the survival rate of mouse bone marrow macrophages at concentrations of 0.1, 0.5, 1 and 3 μM. Treatment of mouse bone marrow macrophages with Platicodin D at 5 and 10 μM concentrations decreased cell viability by 24.92% and 85.8% (IC ₅₀ : 7.06 μM) (FIG. 1 and Table 1). In the examples below, Platicodin D was used at concentrations of 1, 3 and 5 μM that did not significantly affect the survival rate of mouse bone marrow macrophages.

Platicodin D concentration (μM) Absorbance (570 nm) % Survival rate of mouse bone marrow macrophages 0 1.708 100.00 0.1 1.767 103.43 0.5 1.192 111.92 One 2.230 130.52 3 3.052 178.68 5 1.283 75.08 10 0.243 14.20

Example 5 Measurement of Mouse Osteoclast Formation Inhibition by Platicodin D

In order to confirm the inhibitory effect of the platicodine D on RANKL-induced osteoclast formation in mouse bone marrow macrophages, experiments were carried out as described in the literature (Park EK. Et al., Biochem Biophys). Res Commun. , 325 (4): 1472-1480 (2004).

Platicodine D was dissolved in DMSO and then 1, 3 and 5 μM with α-MEM added with 1% antibiotic-antibacterial solution, 10% FBS, RANKL (100 ng / ml) and M-CSF (30 ng / ml). Dilute to a concentration of. Isolated mouse bone marrow macrophages were put into 1 x 10 ⁴ cells per well in a 96-well plate, and cultured by adding 200 μl of α-MEM depending on the diluted concentration. As a control, α-MEM medium solution without Platicodin D was added.

The 96-well plates were exchanged every two days with fresh α-MEM medium added with 1% antibiotic-antibacterial solution, 10% FBS, RANKL (100 ng / ml) and M-CSF (30 ng / ml), at 37 ° C and After culturing for 5 days in a cell incubator at 5% CO ₂ condition, staining was performed using a TRAP assay kit (tartrate resistant acid phosphatase assay kit; Sigma, USA) to confirm the formation of osteoclasts. The number of cells containing three or more nuclei, which are characteristic of osteoclasts, was measured using an optical microscope.

As a result, osteoclasts were not produced in mouse bone marrow macrophages not treated with RANKL, whereas the number of osteoclasts was significantly increased in mouse bone marrow macrophages treated with RANKL (p <0.0001) (FIG. 2 and Table 2). Treatment of mouse bone marrow macrophages with RANKL and Platicodin D did not affect osteoclast formation at 1 μM concentrations, but inhibited RANKL-induced osteoclast formation at 5 and 3 μM concentrations of 55.1 and 93.1%, respectively ( p <0.001). In other words, Platicodin D was confirmed to reduce bone resorption by inhibiting the formation of osteoclasts even at a concentration that does not exhibit cytotoxicity to bone marrow macrophages (Fig. 2 and Table 2).

Platicodin D concentration (μM) Mouse osteoclast count Mouse osteoclast formation inhibition rate (%) 0 364 ± 43 - One 377 ± 53 - 3 163 ± 10 55.13 5 25 ± 6 93.13

Example 6 Measurement of Bone Uptake Inhibitory Activity of Mouse Osteoclasts by Platicodin D

In order to determine the inhibitory effect of platicodine D on bone resorption of RANKL-induced mouse osteoclasts, experiments were performed as described in the literature (Wattel A. et al., J Cell). Biochem. , 92 (2): 285-295 (2004).

Platicodine D was dissolved in DMSO and then 1, 3 and 5 μM with α-MEM added with 1% antibiotic-antibacterial solution, 10% FBS, RANKL (100 ng / ml) and M-CSF (30 ng / ml). Dilute to concentration. In a 12-well plate specially coated with calcium phosphate, 5 x 10 ⁴ macrophages (BMM) isolated from the bone marrow of the 4 weeks old mouse hind limb are placed in each well and α-MEM containing Platicodin D Incubated by adding. As a control, α-MEM medium solution without Platicodin D was added.

Replace 12-well plates with fresh α-MEM medium added with Platicodin D, 1% antibiotic-antibacterial solution, 10% FBS, RANKL (100 ng / ml) and M-CSF (30 ng / ml) every other day The cells were cultured in a cell incubator at 37 ° C. and 5% CO ₂ for 15 days. After culturing the bone marrow macrophages for 15 days, the medium was removed and sodium hypochlorite solution was added. After 5 minutes, the sodium hypochlorite solution was 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 with bone marrow macrophages, production of uptake by osteoclasts was remarkably induced. When RANKL and Platicodin D (1, 3 and 5 μM) were treated together, it was confirmed that the production of uptake by osteoclasts was significantly reduced at concentrations of 3 and 5 μM (FIG. 3). In addition, as in the result of Example 5, bone uptake was markedly suppressed with an increase in the Platicodin D concentration.

Example 7: Determination of Bone Lysis Inhibition by Platicodin D in Human Breast Cancer Induced Osteolytic Animal Model

In order to determine the inhibitory effect of platicodine D on osteolysis of osteoclasts in bone lysis animal models induced by human breast cancer cells, experiments were performed as follows according to the method described in the literature (Julie A). Sharp. Et al., Clinical & Experimental Metastsis, 21: 19-29 (2004).

Six-week-old female Balb / c-nu / nu mice (Central Experimental Animal Co., Ltd., Korea), 6 animals per group, control group, only human breast cells injected, human breast cancer cells and platinum D was divided into groups administered at concentrations of 1 mg / kg and 2 mg / kg (4 groups in total) and purified for a week. To induce bone lysis by human breast cancer cells, mice were anesthetized by intraperitoneal administration with 100 μl of anesthetic (Virbac Laboratories, France): Rumpoon (Bayer Korea Co., Ltd.: PBS = 1: 1: 2). . Breast cancer cells, MDA-MB-231 cells ^{(1 x 10 7 / 100㎕ PBS} ) of the person in the hind leg of the mouse of all groups other than the control mice was injected between the femur and tibia. From the day after the human breast cancer cells were injected into the mouse, Platicodin D was dissolved in PBS and orally administered at 100 μl at 1 mg / kg (body weight) and 2 mg / kg (body weight) for 44 days. 45 days after the administration of Platicodin D, the mice were anesthetized with anesthesia, and the dissolution of the femur in the hind limbs was confirmed by X-ray, and bone density was measured by micro computed tomography (μCT).

As a result, the group injected with only human breast cancer cells showed a marked bone lysis on the X-ray image compared to the control group, and the bone density was reduced by 57.02% compared to the control group. In the group administered orally at 1 mg / kg (body weight) of Platicodin D, the bone density was 43.38%, which did not show the effect of inhibiting osteolysis when compared to the group injected with human breast cancer cells. In the case of oral administration in kg (body weight), bone lysis was confirmed to be 70.66%. In addition, the volume of breast cancer cells injected between the femur and tibia was markedly inhibited by Platicodin D. Therefore, it can be seen that Platicodin D not only inhibits osteoclast formation and bone resorption, but also inhibits the growth of breast cancer cells, thereby reducing osteolytic bone destruction induced by human breast cancer cells (FIGS. 4, 5 and Table 3).

Platicodin D concentration (μM) Bone Mineral Density (%) Bone Density Reduction (%) Control group 100 - Human breast cancer cells
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Platicodine D (0 mg / kg) 42.98 57.02 Human breast cancer cells
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Platicodine D (1 mg / kg) 56.62 43.38 Human breast cancer cells
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Platicodine D (2 mg / kg) 70.66 29.34

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is a graph showing cell viability of mouse bone marrow macrophages by Platicodin D.

2A-2B are results images and graphs showing the inhibitory effect of platicodine D on osteoclast formation.

Figure 3 is an image showing the inhibitory effect of Platicodin D on bone uptake by osteoclasts.

4A-4B are X-ray images and bone density graphs showing the effect of inhibiting osteolysis of Platicodin D in an animal model on bone lysis induced by human breast cancer cells.

Claims (9)

Pharmaceutical composition for preventing and treating bone damage or osteoporosis caused by bone metastasis of cancer cells containing Platicodin D as an active ingredient. The pharmaceutical composition of claim 1, wherein the Platicodin D is contained in an extract of Platycodon grandiflorum . The pharmaceutical composition according to claim 2, wherein the Gil-Gil extract is obtained by treating water, butanol, ethyl acetate, or a combination thereof by Gil-Gil. delete The pharmaceutical composition of claim 1, wherein the composition inhibits the formation of osteoclasts. The pharmaceutical composition of claim 1, wherein the composition inhibits the formation of osteoclasts induced by a receptor activator of NFκB ligand (RANKL). The pharmaceutical composition of claim 1, wherein the composition inhibits bone resorption by osteoclasts. (a) a therapeutically effective amount of Platicodin D; And (b) a pharmaceutical composition for preventing or treating bone damage or osteoporosis caused by bone metastasis of cancer cells comprising a pharmaceutically acceptable carrier. (a) a food effective amount of Platicodin D; And (b) a food composition for improving bone damage or osteoporosis caused by bone metastasis of cancer cells, including a food acceptable carrier.

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