WO2007043810A1

WO2007043810A1 - Compositions comprising the extract of roasted licorice for preventing and treating bone diseases

Info

Publication number: WO2007043810A1
Application number: PCT/KR2006/004100
Authority: WO
Inventors: Won Yoon Chung; Kwang Kyun Park; Sun Kyoung Lee; Soon Sung Lim; Junghan Yoon Park
Original assignee: Industry Academic Cooperation Foundation of Yonsei University
Current assignee: Industry Academic Cooperation Foundation of Yonsei University
Priority date: 2005-10-14
Filing date: 2006-10-12
Publication date: 2007-04-19
Anticipated expiration: 2008-04-14
Also published as: KR100684194B1

Abstract

The present invention relates to a composition for preventing or treating a bone disease which comprises an extract of a roasted licorice as an active ingredient. The present composition prevents the formation of osteoclasts to improve a bone mineral density, particularly effectively inhibiting bone resorption by bone metastasis of cancer cells to exhibit excellent therapeutic and preventive efficacies on bone diseases. Furthermore, the extracts of roasted licorice used as active ingredients in the composition are considered to be a safety ingredient in the senses that they have no toxicity to human and no influence on the viability of bone marrow-derived macrophages.

Description

COMPOSITIONS COMPRISING THE EXTRACT OF ROASTED LICORICE FOR

PREVENTING AND TREATING BONE DISEASES

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to a composition for preventing and treating bone diseases comprising the extract of a roasted licorice as an active ingredient.

DESCRIPTION OF THE RELATED ART Bone supports human soft tissues and body weight and protects inner organs such as intestines from outer impacts and shocks by surrounding inner organs. In addition to such supporting functions for muscle and intestines, it has an important function as a reservoir for essential body mineral components such as calcium, phosphorous and magnesium. Adult bones with terminated growth are not static, and undergo a dynamic and continuous rebuilding process in which resorption and new bone formation occur. This process is called as bone remodeling (Yamaguchi A. et al., Tanpakushitsu Kakusan Koso., 50(6Suppl):664-669(2005)). The bone turnover, a process in which old bones are removed and replaced with new bones, is pivotal for the repair of micro-damage of bones associated with growth and stress and the maintenance of bone functions (Cohen-Solal M. et al., Therapie., 58(5):391- 393(2003)).

Bone remodeling has been reported to involve two types of cells. One of them is bone-forming osteoblasts and the other is bone-resorbing osteoclasts. Osteoblasts produce RANKL (receptor activator of nuclear factor-κB ligand) and its decoy receptor, OPG (osteoprotegerin). RANKL binds to RANK (receptor activator of nuclear factor- KB) on osteoclast progenitor cells that are then maturated to osteoclasts, finally inducing bone-resorption. However, where OPG binds to RANKL to block the binding between RANKL and RANK, the formation of osteoclasts is inhibited and bone- resorption does not occur at undesirable level (Theill LE. et al., Annu Rev Immunol.,

20:795-823(2002); Wagner EF. et al., Curr Opin Genet Dev., 11:527-532(2001)). Resorption or disruption of old bones is induced by osteoclasts generated in blood cells (hematopoietic cells), that allows a trace amount of calcium ions to be released into blood circulation by forming pores on bones to contribute to mineral homeostasis (William J. et al., Nature., 423:337-342(2003)).

Osteoblasts produced in bone cells are involved in the filling of pores with collagen and covering of pores with precipitates of calcium and phosphorous (hydroxyapatite), thereby forming new bones to rebuild skeleton (Stains JP. et al., Birth Defects Res C Embryo Today., 75(l):72-80(2005)). It takes about 100 days to disrupt old bones and rebuild new bones (Schwarz EM. et al., Curr Opin Orthop., 11:329-335(2000)). While 100% of calcium content in bone is changed within 1 year in an infant, about 10-30% of the skeleton is rebuilt by the bone remodeling in an adult. Only if the two processes, resorption and bone formation are quantitatively equal, the bone mass can be maintained. Where such remodeling process becomes unbalanced, a variety of diseases or disorders such as osteoporosis and damages associated with bone metastasis of cancer cells are caused.

Breast cancer, prostate cancer and multiple myeloma are accompanied with bone metastasis (Kozlow W. et al., J Mammary Gland Biol Neoplasia., 10(2): 169- 180(2005)). The life span of patients having such cancer has been suggested to be dependent on bone metastasis.

Breast cancer is one of the most prevalent cancers developed in women in advanced nations such as USA and Europe and is the first death cause of American women aged 40-55. The incidence of breast cancer is about 1 person/9 women, and the number of breast cancer patients becomes increasing by about 15% rate. Breast cancer is also the most predominant one for Korean women and its incidence is expected to be increasing. Prostate cancer is also one of prevalent cancers and accounts for 20% of the death of man cancer patients in USA or Europe. In addition, the incidence of prostate cancer in Korea becomes sharply increasing because of westernized dietary life and switching over to aging society. The reason why the mortality of patients having breast or prostate cancer becomes elevated is that cancer cells are selectively metastasized into bones. The bone metastasis has been found in eighty percentages of breast cancer patients and causes osteolysis due to metastasized breast cancer cells, eventually resulting in skeletal fractures. In addition, the bone metastasis causes various bone-related diseases such as leukoerythroblastic anaemia, bone deformity, hypercalcemia, pain and nerve- compression syndromes (Roodman GD., N Engl J Med., 350:1655-1664(2004)). The bone metastasis observed in breast cancer is almost an osteolytic metastasis leading to bone resorption in which breast cancer cells do not affect directly bone but stimulates osteoclasts (Boyde A. et al., Scan Electron Microsc, 4:1537-1554(1986)). Unlikely, the bone metastasis found in prostate cancer is an osteoblastic metastasis. The osteoblastic metastasis has been also reported to relate directly to osteolysis.

Events and phenomena of the initial phase in bone metastasis are similar to those of metastases to other organs. The proliferation, migration and invasion of cancer cells, degradation of extracellular matrix and angiogenesis occur to deliver cancer cells to capillary beds in bone through circulation. The delivered cancer cells adhere to vascular endothelial cells and escape the blood vessel, eventually entering bones. The cancer cells entering bones proliferate in bone-surrounding microenvironments and stimulate the activity of osteoclasts or osteoblasts, thereby determining whether the subsequent bone metastasis is osteolytic or osteoblastic (Choong PF. et al., Clin Orthop Relat Res., 415S:S19-S31(2003)). Cancer cells positioned in bone secrete osteolytic factors such as PTHrP (parathyroid hormone- related protein), IL (interleukin)-l, IL-6, IL-8 and IL-Il (Bendre M., et al., Clin Orthop Relat Res., 415 Suppl:S39-S45(2003); Palmqvist P. et al., J Immunol., 169(6):3353-3362(2002)). The secreted factors down-regulate the expression of OPG in osteoblasts and up-regulate the expression of RANKL. RANKL increasingly expressed binds to RANK of osteoclast progenitor cells and to permit the maturation of a multitude of osteoclast progenitor cells, thereby inducing bone destruction by excessive bone resorption. The excessive bone resorption leads to the release of various growth factors from bone matrix such as TGF (transforming growth factor)-β, IGF (insulin-like growth factor)-I and II, and FGF (fibroblast growth factor)-I and II (Yin JJ. et al, Cell Res., 15(l):57-62(2005)), responsible for growth and proliferation of breast cancer cells (Pfeilschifter J. et al., Proc Natl Acad Sci U S A., 84(7):2024- 2028(1987)). Bone metastasis of cancer cells proceeds with the vicious cycle described above (Gregory R., Nat Rev Cancer, 2:584-593(2002)).

Osteoporosis is defined as a disease characterized by low bone mass and deterioration of bone microstructure, causing bone fragility and increased risk of fracture. The disease is developed by unbalanced remodeling exhibiting osteoclastic activities higher than osteoblastic activities (Iqbal MM., South Med 1, 93(1): 2- 18(2000)).

While the inner structure of normal bones has a compact network, the osteoporosis bone shows a widened space between structures and a thinner micro- architecture that becomes susceptible to skeletal fractures (Stepan JJ. et al., Endocr ReguL, 37(4):225-238(2003)). Osteoporosis is classified into postmenopausal osteoporosis in which the bone loss (2-3% a year) appears upon initiation of menopause and the risk of spine compression and wrist bone fracture is elevated; senile osteoporosis in which it is developed slowly (0.5-1% a year) in elder men and women aged more than 70 years and induces gradual bone loss of hip and spine bones; and secondary osteoporosis developed by diseases {e.g., endocrine diseases, gastrointestinal diseases and cancer), drugs {e.g., adrenal cortical hormones, anticancer chemotherapy, thyroid hormones, anticonvulsants, antiplatelets, methotexate, cyclosporine and GnRH), alcohol, smoking or accidents (Rosen CJ., N Engl J Med., 353(6):595-603(2005); Davidson M., Clinicain Reviews., 12(4):75-

82(2002)).

COX (cyclooxygenase) is a main enzyme involved in the biosyntheis of prostaglandins (PGs) (Vane JR. et al., Annu Rev Pharmacol Toxicol., 38:197- 120(1998)), including two isozymes COX-I and COX-2. COX-I constitutively exists in stomach and kidney and is involved in homeostasis, whereas COX-2 is temporarily and rapidly expressed upon the development of diseases by mitogens and cytokines.

It has been reported that osteoblast-generated prostaglandin E₂ (PGE₂) induced by the interaction between cancer cells and osteoblasts as well as osteolytic factors such as cancer cell-produced cytokines promotes bone resorption and is related closely to rheumatoid arthritis and postmenopausal osteoporosis (Sugiyama TJ., Bone Miner. Metab., 19:89-96(2001)). PGE₂ is produced from arachidonic acid in osteoblasts by COX-2 (cyclooxygenase-2) regulated by NF(nuclear factor)-κB pathway and MAP kinase (mitogen-activated protein kinase), and binds to EP (E series of PG receptor) 4 to induce the expression of RANKL, eventually leading to osteoclastogenesis and bone resorption (Miyaura C, et al., J Biol Chem., 275:19819- 19823(2000); Suzawa T. et al., Endocrinology, 141:1554-1559(2000)). Furthermore, PGE₂ inhibits secretion of OPG in osteoblasts and up-regulates expression of RANK in osteoclasts (Liu X. et al., Endocrinology, 146:1991-1998(2005)). It has been suggested that inhibitors to PGs biosynthesis are effective in the blocking of osteolysis due to cancer cells (Seyberth HW. et al., N Eng J Med., 293:1278- 1283(1975)) and COX-2 knockout mice show reduced bone resorption responsive to PTH (parathyroid hormone) or 1,25-dihydroxyvitamin D₃ (Okada Y. et al., J Clin Invest, 105:823-832(2000)). Taken together, it could be understood that inhibitors to COX-2 reduce the activation of osteoclasts to inhibit bone resorption, finally successfully preventing and treating bone diseases.

Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THIS INVETNION

The present inventors have made intensive researches to develop a novel substance with safety, plants-originated substances, for effectively preventing or treating bone diseases. As a result, we have discovered that a roasted licorice having been used as oriental medicines is very effective in preventing or treating bone diseases, eventually accomplishing the present invention.

Accordingly, it is an object of this invention to provide a composition for preventing or treating bone diseases.

It is another object of this invention to provide a method for preventing or treating bone diseases.

It is still another object of this invention to provide a novel use for manufacturing a medicament for preventing or treating bone diseases.

Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.

In one aspect of this invention, there is provided a composition for preventing or treating a bone disease, which comprises an extract of a roasted licorice as an active ingredient.

In another aspect of this invention, there is provided a method for preventing or treating a bone disease, which comprises administering to a subject a composition comprising an extract of a roasted licorice as an active ingredient. In still another aspect of this invention, there is provided a use of a roasted licorice for manufacturing a medicament for preventing or treating bone diseases.

The present inventors have made intensive researches to develop a novel substance with safety, plants-originated substances, for effectively preventing or treating bone diseases. As a result, we have discovered that a roasted licorice having been used as oriental medicines is very effective in preventing or treating bone diseases.

The term used herein "licorice" refers to a perennial plant belonging to Leguminosae including Glycyrrhiza uralensis Fisch, Glycyrrhiza glabra L, Glycyrrhiza glabra and Glycyrrhiza pallid/flora, preferably, Glycyrrhiza uralensis Fisch or

Glycyrrhiza glabra L. According to a preferred embodiment, the term "licorice" refers to a dried root or rootstock of licorice.

The most prominent feature of this invention is to use extracts from roasted licorice rather than licorice as active ingredients. The term used herein "roasted licorice" refers to a licorice processed by heating. The preparation of roasted licorices may be carried out according to various processes. According to conventional preparations, licorices are wrapped with wet straw ropes and then buried in a fire turning to ashes to give roasted licorices. Recently, licorices are wrapped with paper and then wet paper, and roasted with fires to prepare roasted licorices. The roasted licorice used in this invention includes all products of roasted licorices commercially available and used in the field of oriental medicine.

According to a preferred embodiment, the roasted licorice is prepared by indirect contact {e.g., heating in vessels) of fires of 150-250⁰C to dried licorices. At this time, the color of licorices is changed from yellow to maroon.

Compared with non-heated licorice, the roasted licorice prepared by heating contains larger contents of nonpolar compounds with biological activities such as licochalcone A and smaller contents of isoliquiritigenin and glycyrrhizin (see Experimental Example 1). The change in biologically active ingredients is responsible for excellent efficacies of roasted licorices on treating or preventing bone diseases.

The "roasted licorice extract" used as active ingredients in this invention means extraction products from roasted licorices using conventional extraction solvents. Preferably, the roasted licorice extract of this invention is obtained from roasted licorices using (a) water, (b) absolute or water-bearing lower alcohol containing 1-4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) mixture of lower alcohol and water, (d) acetone, (e) ethyl acetate, (f) chloroform, (g) butylacetate, (h) 1,3-butylene glycol, (i) hexane or (j) diethylether. It is apparent to one skilled in the art that other conventional solvents may be employed for obtaining roasted licorice extracts having substantially identical effects.

More preferably, the present extraction is prepared by extracting the roasted licorice with water, methanol, ethanol or their combinations, most preferably, ethanol or mixture of water and ethanol. The extracts of this invention include those subject to additional purification by the well-known methods in the art as well as those obtained by extraction. For instance, it could be appreciated that active fractions obtained using a variety of additional purification methods such as an ultra-filtration with defined molecular weight cut-off value and various chromatography (designed for purification dependent upon size, charge, hydrophobicity and affinity) are included in the present extracts.

The extracts of this invention may be obtained in the form of powder by use of vacuum distillation, lyophilization or spray drying.

The present composition for preventing or treating bone diseases inhibits the expression of COX (cyclooxygenase)-2 and RANKL (receptor activator of nuclear factor-κB ligand) and promotes the expression of OPG (osteoprotegerin) in osteoblasts to prevent the binding between RANKL of osteoblasts and RANK of osteoclast progenitor cells, which inhibits the differentiation of osteoclast progenitor cells into osteoclasts to prevent the formation of osteoclasts, eventually resulting in the improvement in bone mineral density and successful treatment or prevention of various bone diseases.

Examples of bone diseases prevented or treated by the present composition include, but not limited to, low bone mass, osteoporosis, bone fracture, bone refracture, bone defect, osteomalacia, Behcet's syndrome in bone, Paget's disease, rigid myelitis, rheumatoid arthritis, osteoarthritis, bone loss, achondroplasia, osteochondritis, hyperparathyroidism, osteogenesis imperfecta, congenital hypophosphatasia, fibromatous lesions, fibrous displasia, multiple myeloma, abnormal bone turnover, osteolytic bone disease and periodontal disease. Furthermore, exemplified bone diseases prevented or treated by the present composition include bone diseases associated with post-traumatic bone surgery, post-plastic bone surgery, bone chemotherapy treatment, bone radiotherapy treatment and bone metastasis of cancer cells {e.g., breast cancer cells). According to a preferred embodiment, bone diseases prevented or treated by the present composition include bone damages associated with bone metastasis of cancer cells, osteoporosis, osteomalacia, rickets, osteitis fibrosa, aplastic bone disease, metabolic bone disease, osteolysis, leucopenia, bone deformation, hypercalcemia and nerve compression. The composition of the present invention may be formulated to provide a pharmaceutical composition or a food composition.

The pharmaceutical composition of this invention comprises (i) a therapeutically effective amount of an extract of a roasted licorice; and (b) a pharmaceutically acceptable carrier. The term used herein "therapeutically effective amount" refers to the amount sufficient to exhibit therapeutic efficacies described above. For example, the amount of the roasted licorice extract is 0.1-90 wt% in the pharmaceutical composition of this invention. The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention, which is commonly used in pharmaceutical formulations, but is not limited to, includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils. The pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's

Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

A pharmaceutical composition of this invention may be administered orally or parenterally. For non-oral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection and transdermal administration may be employed.

The correct dosage of the pharmaceutical compositions of this invention will be varied according to the particular formulation, the mode of application, age, body weight and sex of the patient, diet, administration time, administration route, excretion rate and reaction sensitivities. It is understood that the ordinary skilled physician will readily be able to determine and prescribe a correct dosage of this pharmaceutical compositions. For instance, the daily dosage of the present pharmaceutical composition may be in the range of 0.01 mg/kg (body weight) to 2 g/kg (body weight). The administration may be once or several times a day.

According to the conventional techniques known to those skilled in the art, the pharmaceutical compositions of this invention can be formulated with pharmaceutical acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dosage form. Non-limiting examples of the formulations include, but not limited to, a solution, a suspension or an emulsion, an extract, an elixir, a powder, a granule, a tablet and a capsule.

The present composition for preventing or treating bone diseases may be prepared to provide a food composition, in particular, a health food composition. The food composition may comprise conventional additives for preparing food compositions, e.g., proteins, carbohydrates, lipids, nutritive substances and flavors. For example, where the food composition of this invention is provided as a drink, it may further comprise flavors and natural carbohydrates as well as lactic acid bacteria and collagen as active ingredients. Non-limiting examples of natural carbohydrates include, but not limited to, monosaccharide {e.g., glucose and fructose), disaccharide {e.g., maltose and sucrose), oligosaccharide, polysaccharide {e.g., dextrin and cyclodextrin) and sugar alcohol {e.g., xylitol, sorbitol and erythritol). Non-limiting examples of flavors include, but not limited to, natural flavors {e.g., thaumatin and extract of Stevia) and synthetic flavors {e.g., saccharin and aspartame). Given the availability to foods, the food composition of this invention is very useful in preventing or treating bone diseases.

As described hereinabove, the present composition inhibits the formation of osteoclasts to highly improve bone mineral density, in particular, effectively preventing bone resorption by bone metastasis of cancer cells to exhibit enhanced therapeutic and preventive efficacies on bone diseases. In these connections, the present composition may be also defined as "compositions for inhibiting the formation of osteoclasts", "compositions for improving bone mineral density" and "compositions for preventing bone resorption by bone metastasis of cancer cells." The extracts of roasted licorices used as active ingredients in the composition are considered to be a safety ingredient in the senses that they have no toxicity to human and no influence on the viability of bone marrow macrophage. The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

BRIEF DESCRIPTION OF THE DRAWINGS

Hg. 1 represents HPLC (high performance liquid chromatography) analysis results of ethanol extracts of licorice and roasted licorice.

Fig 2 is a graph representing a standard curve for glycyrrhizin quantification. Rg 3 is a graph representing a standard curve for isoliquiritigenin quantification.

Rg. 4 is a graph representing a standard curve for licochalcone A quantification.

Hg. 5 represents effects of roasted licorice extract on the expressions of RANKL and OPG mRNA in osteoblasts treated with conditioned media (CM) of the human metastatic breast cancer cell line, MDA-MB-231.

Fig. 6 represents effects of roasted licorice extract on the expression of the RANKL protein in osteoblasts treated with conditioned media (CM) of MDA-MB-231 cells. Rg. 7 represents effects of roasted licorice extract on the expression of COX mRNA in osteoblasts treated with conditioned media (CM) of MDA-MB-231 cells.

Fig. 8 represents effects of roasted licorice extract on the expression of the COX-2 protein in osteoblasts treated with conditioned media (CM) of MDA-MB-231 cells. Fig. 9 represents experimental results to evaluate whether roasted licorice extracts show cytotoxicity to mouse bone marrow-derived macrophages.

Figs. 10a- 10b represents effects of roasted licorice extract on the formation of RANKL-induced osteoclasts in mouse bone marrow-derived macrophages. Fig. 11 represents effects of roasted licorice extract on the formation of resorption pit by osteoclasts.

Figs 12a-12b represents comparative analyses results for effects of licorice and roasted licorice extracts on the formation of RANKL-induced osteoclasts in mouse bone marrow-derived macrophages.

Fig. 13 represents comparative analyses results for effects of licorice and roasted licorice extracts on the formation of resorption pit by osteoclasts.

Fig. 14 represents CT images to show the inhibitory effects of roasted licorice extracts on bone metastasis of breast cancer cells in animal models. Arrows denote bone damages caused by cancer-induced osteoclasts.

EXAMPLES EXAMPLE 1: Preparation of Extract of Roasted Licorice Using Ethanol

Dried licorice purchased from the Gyeondong Market (Korea) was additionally dried under shady environments for about 5 days at room temperature and pulverized. The pulverized licorice was placed into an iron kettle and roasted for 20 min at 150-250⁰C to become maroon color from yellow color, followed by the extraction using 95% ethanol as extraction solvents. For extraction, either the machinery extraction (ASE300 ACCELERATED SOLVENT EXTRACOR) or ultrasonic extraction (BRANSON Ultrasonics Corporation) was employed. In the machinery extraction, 300 ml of 95% ethanol were added to 30 g of the pulverized licorice and the extraction was subsequently carried out 1500 psi for 20 min at 50⁰C . In the ultrasonic extraction, 1 L of 95% ethanol was added to 100 g of the pulverized licorice and the extraction was subsequently carried out for 2-3 weeks at 50⁰C . The extracted samples were then concentrated under reduced pressures at 45^°C

(Biotron corporation, Modul spin 40) to give samples for experiments.

COMPARATIVE EXAMPLE 2: Preparation of Extract of Licorice Using Ethanol

The ethanol extract from licorice was prepared in accordance with the same manner as Example 1, except that non-roasted licorice was used.

REFERENCE EXAMPLE 1: Statistical Analysis

The experimental results were represented as mean ± standard deviation (SD). Student t-test was employed for statistical analyses. If p value is less than 0.05, it is determined to be statistically significant.

EXPERIMENTAL EXAMPLE 1: Comparison of Ingredients of Extracts from Licorice and Roasted Licorice

The ethanol extracts of licorice and roasted licorice were analyzed under HPLC conditions of Table 1 for comparison (Table 1 and Fig. 1). TABLE 1

The extract of roasted licorice was analyzed to contain increased contents of licochalcone A and a group of nonpolar compounds comprising licochalcone A and decreased contents of isoliquiritigenin and glycyrrhizin (Table 2). The quantities of ingredients contained in extracts of licorice or roasted licorice were determined using standard curves (Figs. 2-4). TABLE 2. Contents of Ingredients in Extracts of Licorice or Roasted Licorice

EXPERIMENTAL EXAMPLE 2: Effects of Roasted Licorice Extract on Expressions of RANKL and OPG mRNA in Human Osteoblast Cells (hFOBl.19 Cells) Treated with Conditioned Media of Human Metastatic Breast Cancer Cells

2-1. Culture of Breast Cancer Cells and Preparation of Conditioned Media

Human metastatic breast cancer cell line, MDA-MB-231 cells (1 x 10⁶ cells) were cultured for 3 days at 37⁰C in L-15 (Leibovitz's-15) media supplemented with 1% antibiotic-antimycotic solution (100 units/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B) and 10% fetal bovine serum (FBS). The culture was conducted to 80-90% confluent, the media was then discarded, and the cells were washed twice with phosphate buffered-saline (PBS), followed by additional 24-hr culturing in DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F- 12(Ham) = 1:1) with no FBS. The culture media were centrifuged at 1700 rpm for 5 min at 4°C to remove cells and then supernatants containing various osteolytic factors secreted by MDA-MB-231 cells were collected. Before experiments, supernatants were stored at -20 ^°C .

2-2. Culture of Human Osteoblast Cells, hFOBl.19

Human osteoblast cells, hFOBl.19 cells (1 x 10⁶ cells) were cultured in DMEM/F12 medium supplemented with 1% antibiotic-antimycotic solution (100 units/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B) and 10% FBS. 2-3. Effects of Roasted Licorice Extract on Expressions of RANKL and OPG mRNA in hFOBl.19 Cells

The expressions of RANKL and OPG mRNAs in human osteoblast hF0B1.19 cells were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). hF0B1.19 cells (1 x 10⁵ cells) were cultured in 10% FBS-DMEM/F12 media.

After reaching 80-90% confluent, the media were changed with differentiation media (DM) (DMEM/F12 supplemented with 50 μg/ml Vitamin C, 10^~8 M vitamin D₃, 10^"8 M Vitamin K₃, 10% charcoal -stripped FBS) and the culture was carried out for 3 days for differentiation to mature osteoblasts. After removing DM, cells were cultured for 6 hr in DM media containing 0, 2.5, 5.0, 7.5 or 10 μg/ml of roasted licorice extract and CM of MDA-MB-231 cells. Total RNAs were isolated using TRIZOL reagent (Life Technologies, Grand Island, NY, USA) from collected osteoblasts. The reverse transcription was performed to synthesize cDNA using reaction mixtures (20 μl) containing isolated ¹ RNA (2 μg), oligo-dTi₅ (0.5 μg), ribonuclease inhibitor and reverse transcriptase. The amplifications of cDNA were performed using PCR reactions containing cDNA (1 μg), primers (10 pmol), dNTP (250 μM), MgCI₂ (1.5 mM) and Taq DNA polymerase (1.5 units). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was employed as control. The primer sets used for PCR were as follows: RANKL (665 bp): sense, 5'- ATAGAATATCAGAAGATGGCACTC-S', antisense, S'-TAAGGAGGGGTTGGAGACCTCG-

3'; OPG (409 bp): sense 5'-GGG GACCACAATGAACAAGTTG-S¹, antisense 5'- AGCTTGCACCACTCCAAATCC-3'; GAPDH (420 bp): sense 5'- CCGCCTACTGCCCACTGCCACCAC-S¹, antisense 5'-

TCCATCCACTATGTCAGCAGGTCC-3'. The PCR reactions were conducted under the following thermal conditions: 35 cycles of 1 min at 94°C (denaturation), 1 min at annealing temperatures (annealing) and 1 min at 72⁰C (extension). The annealing temperatures were set to be 62°C (RANKL), 55°C (OPG) and 52°C (GAPDH). The PCR products were detected on 2% agarose gel, stained with ethidium bromide and observed using UV transilluminator. The band intensities of RANKL, OPG and GAPDH were measured using the image analysis software (ΗNA, version 2.0) and normalized with GAPDH. The ratio of RANKL/OPG was investigated. 2-4. Results

Osteoblasts treated with CM of MDA-MB-231 cells showed increased expression of RANKL mRNA and decreased expression of OPG mRNA; therefore, the ratio of RANKL/OPG was elevated. In contrast, cells treated with roasted licorice extract together with CM of MDA-MB-231 cells showed decreased expression of RANKL mRNA and increased expression of OPG mRNA; therefore, the ratio of RANKL/OPG was reduced. These expression profiles were dependent on the amount of roasted licorice extracts. Accordingly, it could be appreciated that the roasted licorice extract down-regulates the expression of RANKL increased by CM of MDA- MB-231 cells and up-regulates the expression of OPG decreased by CM to lower the binding between RANK of osteoclast progenitor cells and RANKL of osteoblasts, inhibiting bone-resorption by osteoclasts (Fig. 5).

EXPERIMENTAL EXAMPLE 3: Inhibitory Effects of Roasted Licorice Extract on Expressions of RANKL Induced by CM of MDA-MB-231 Cells in hFOB1.19 Cells

3-1. Experimental Methods hF0B1.19 cells (1 x 10⁵ cells) were cultured in 10% FBS-DMEM/F12 media. After reaching 80-90% confluency, the media were changed with differentiation media (DM) and the culture was carried out for 3 days for differentiation to mature osteoblasts. After removing DM, cells were cultured for 48 hr in DM media containing 0, 2.5, 5.0, 7.5 or 10 μg/ml of roasted licorice extract and CM of MDA-MB-231 cells. Cells were washed using PBS and incubated with 0.25% trypsin-EDTA solution. Cells adhered to culture dishes were collected into a tube and centrifuged at 1500 rpm for 3 min. hFOB1.19 cells were suspended in PBS containing 1% BSA and centrifuged at

1500 rpm for 5 min. hFOB1.19 cells were resuspended in PBS containing 1% BSA and their aliquots (2 x 10⁵ cells) were transferred into 1.5 ml eppendorff tubes. Cells were incubated over ice for 30 min with a primary antibody against RANKL (monoclonal anti-human TRANCE/TNFSF11 antibody; R&D System Inc., MN, USA) and 1 ml of PBS containing 1% BSA was added, which after centrifugation was performed at 1500 rpm for 10 min. After discarding supernatant, cells were incubated over ice for 30 min with FITC (fluorescein isothiocyanate)-conjugated secondary antibody, goat anti-mouse IgG (Southern Biotechnology Associates Inc., Birmingham, AL, USA). 1 ml of PBS containing 1% BSA was added into tubes and centrifugation at 1500 rpm for 10 min was performed, followed by discarding supernatant. 400 μl of 1% BSA-PBS were mixed well with cells and the cell suspension was transferred to tubes for flow cytometry; FACS). The expression profiles of the RANKL protein on osteoclasts was measured using flow cytometry.

3-2. Results

We investigated whether the extracts of roasted licorice permit to inhibit the increase of the RANKL protein in hFOB1.19 cell induced by CM of MDA-MB-231 cells. Where hFOB1.19 cells were incubated with CM of MDA-MB-231 cells, the expression of RANKL protein was increased. In contrast, hFOB1.19 cells incubated with the extracts of roasted licorice along with CM of MDA-MB-231 cells exhibited lowered expression of the RANKL protein in a dose-dependent manner (Fig. 6 and Table 3). These results urge us to reason that the extract of roasted licorice inhibits RANKL expression in osteoblasts and decreases the binding between RANK of osteoclast progenitor cells and RANKL of osteoblasts to prevents the formation of mature osteoclasts, thereby blocking bone-resorption by osteoclasts. TABLE 3

EXPERIMENTAL EXAMPLE 4: Effects of Roasted Licorice Extract on Expressions of Cyclooxygenase (COX) Induced by CM of MDA-MB-231 cells in Osteoblasts

4-1. Cell Culture hF0B1.19 cells (1 x 10⁶ cells) were cultured in 10% FBS-DMEM/F12 media. After reaching 80-90% confluent, the media were changed with differentiation media (DM) and the culture was carried out for 3 days for differentiation to mature osteoblasts. After removing DM, cells were cultured in media containing 0, 2.5, 5.0, 7.5 or 10 μg/ml of roasted licorice extract or CM of MDA-MB-231 cells either for 1 hr to evaluate the expression of the COX mRNA or for 6 hr to evaluate the expression of the COX protein.

4-2. COXmRNA Expression To evaluate the profile of mRNA expression, total RNA were isolated using

TRIZOL reagent from collected hF0B1.19 cells. The reverse transcription was performed to synthesize cDNA using reaction mixtures (20 μl) containing isolated RNA (2 μg), oligo-dT₁₅ (0.5 μg), ribonuclease inhibitor and reverse transcriptase. The amplifications of cDNA were performed using PCR reactions containing cDNA (1 μg), primers (10 pmol), dNTP (250 μM), MgCl₂ (1.5 mM) and Taq DNA polymerase

(1.5 units). GAPDH was employed as control. The primer sets used for PCR were as follows: GAPDH (420 bp): sense, 5'-CCGCCTACTGCCCACTGCCACCAC-3', antisense, 5'-TCCATCCACTATGTCAGCAGGTCC-S'; COX-I (304 bp): sense, 5'- CATCCTCGACGGCATCTCAGC-3', antisense, 5'-TTGGGTCAGGGGTGGTTATTG-3^I; COX-2 (305 bp): sense, S'-ATGACTTCCAAGCTGGCCGT-S', antisense, 5'-

CCTCTTCAAAAACTTCTCCACACC-3'.

The PCR reactions were conducted under the following thermal conditions: 35 cycles of 1 min at 94⁰C (denaturation), 1 min at annealing temperatures (annealing) and 1 min at 72°C (extension). The annealing temperatures were set to be 63°C (COX-I), 57.5°C (COX-2) and 52⁰C (GAPDH). The PCR products were electrophoresed on 2% agarose gel, stained with ethidium bromide and observed using UV transilluminator. The band intensities of COX-I and COX-2 were normalized with GAPDH.

4-3. COX Protein Expression

Following the collection of hF0B1.19 cells cultured for 6 hr, a cell lysis solution containing 5OmM Tris-HCI, 5mM EDTA, 10% glycerol, 0.1% SDS, 0.2% Triton X-100, 5 μg/ml aprotinin, 1 mM PMSF and protease inhibitor cocktail tablet (Roche, Penzberg, Germany) was incubated with hF0B1.19 cells to isolate proteins. After quantifying proteins, 40 μg of proteins were electrophoresed and transferred to PVDF (polyvinylidene fluoride) membranes, followed by blocking with 5% skim milk. The membranes washed three times for 5 min with TBST (0.1% Tween 20- containing Tris-buffered saline) and incubated with a primary antibody (polyclonal anti-human COX-2 antibody; Cayman Chemical, MI, USA and polyclonal anti-human actin antibody; Sigma, MO, USA). After washing with TBST, the resultant was incubated with a secondary antibody, horseradish peroxidase-conjugated goat anti- rabbit IgG (Santa Cruz Biotechnology, CA, USA). After washing three times for 10 min with TBST, the expression of the COX-2 protein was observed by exposure to X- ray films using ECL kit (enhanced chemiluminescence kit; Santa Cruz Biotechnology, CA, USA).

4-4. Results hFOB1.19 cells treated with CM of MDA-MB-231 cells containing osteolytic factors such as ILs and PTHrP exhibited the increased expression of COX-2 mRNA but unchanged expression of COX-I mRNA. In contrast, cells treated with roasted licorice extract together with CM of MDA-MB-231 cells exhibited the dramatic decrease in COX-2 mRNA expression but unchanged expression of COX-I mRNA (Fig. 7). Such expression pattern of COX-2 mRNA was dependent on doses of roasted licorice extracts (Fig. 8). Accordingly, it could be recognized that the roasted licorice extract down-regulates the expression of COX-2 increased by media of cancer cells and in turn inhibits the expression of RANKL to prevent the formation of osteoclasts, contributing to the inhibition of bone-resorption.

EXPERIMENTAL EXAMPLE 5: Cytotoxicity of Roasted Licorice Extract to Mouse Bone Marrow-derived Macrophage

5-1. Isolation of mouse bone marrow-derived macrophage (BMM) cells 4-week male ICR mice were sacrificed by cervical dislocation and then the outer skin of a hind limb was removed. The hind limb was dissected using surgical scissors and immersed in α-MEM (Minimum Essential Medium Alpha) with no serum.

Bones in muscles were isolated using forceps and transferred into fresh α-MEM. A needle of 1-ml syringe, charged with 500 μl α-MEM was inserted into bone marrow of the hind limb and bone marrow cells were then extracted by spraying. BMM cells were isolated from extracted bone marrow cells using Histopaque (Sigma, MO, USA).

5-2. Evaluation on Cytotoxicity of Roasted Licorice Extract to BMM cells

BMM cells (1 x 10⁴ cells) were aliquot to each well of 96-well plates and cultured in α-MEM supplemented with 1% antibiotic-antimycotic solution, 10% FBS,

M-CSF (macrophage- colony stimulating factor) 30 ng/ml and roasted licorice extract

(0, 2.5, 5.0 and 7.5 μg/ml). The samples of roasted licorice extracts were prepared by dissolving in DMSO (dimethyl sulfoxide) and diluting with 10% FBS-α-MEM to concentrations of 0, 2.5, 5.0 and 7.5 μg/ml. Cells were cultured with changing media every 2 days for 5 days at 37°C in a 5% CO₂ incubator and then were incubated with 5 mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). After 4-hr culture at 37°C, the media and MTT solution were discarded and 200 μl of DMSO were added to each well. 20 min later, the absorbance values at 570 nm were measured. The cell viability was calculated by the percentage ratio of absorbance of cells treated with roasted licorice extract to absorbance of control.

5-3. Results

It was evaluated that roasted licorice extracts at concentrations of 0-7.5 μg/ml have no influence on cell viability of BMM cells (Rg. 9 and Table 4). TABLE 4

EXPERIMENTAL EXAMPLE 6: Inhibitory Effect of Roasted Licorice Extract on Formation of Osteoclasts Induced by RANKL in BMM cells

6-1. Methods

BMM cells were aliquoted with IxIO⁴ CeIIs per each well of 96-well plates and incubated with 10% FBS-α-MEM supplemented with RANKL (100 ng/ml), M-CSF (30 ng/ml) and roasted licorice extract (0, 2.5, 5.0, 7.5 μg/ml). Control was treated solely with 10% FBS-α-MEM. Following 5-day incubation, the formation of osteoclasts was examined by staining with TRAP analysis kit (tartrate resistant acid phosphatase assay kit; Sigma, MO, USA). Cells having nuclei of more than 3, which is characteristics of osteoclasts, were counted under an optical microscope. 6-2. Results

Only 1-2 osteoclasts were observed in BMM cells not treated with RANKL; however, the significantly increasing number of osteoclasts was found in BMM cells treated with RANKL (p <0.0001) (Rg. 10a). The treatment of roasted licorice extracts was shown to decrease the formation of osteoclasts induced by RANKL in a dose-dependent manner (roasted licorice extract 5.0 μg/ml, p < 0.01; 7.5 μg/ml, p < 0.001) (Figs. 10a and 10b, Table 5). TABLE 5

EXPERIMENTAL EXAMPLE 7: Inhibitory Effect of Roasted Licorice Extract on Bone Resorption by Osteoclasts

7-1. Methods

BMM cells of a hind limb of 4-week mice were aliquot with 5 x 10⁴ cells per each well of 24-well plates coated with calcium phosphate. 10% FBS-α-MEM supplemented with RANKL (100 ng/ml), M-CSF (30 ng/ml) and roasted licorice extract (0, 2.5, 5.0, 7.5 μg/ml) was added to each well and culture was performed for 15 days with changing media with a fresh medium every 2 days. Media were discarded and sodium hypochlorite was incubated with cells. 5-min later, the sodium hypochlorite solution was removed and plate was washed twice with distilled water. The formation of resorption pits by osteoclasts was observed under an optical microscope.

7-2. Results We examined the effect of roasted licorice extracts on the formation of resorption pits by osteoclasts induced with RANKL. Roasted licorice extracts were revealed to considerably inhibit the formation of resorption pits in a dose-dependent manner (Fig. 11).

EXPERIMENTAL EXAMPLE 8: Comparative Studies on Effects of Licorice and Roasted Licorice Extracts

8-1. Comparative Analyses of Inhibitory Effects of Licorice and Roasted Licorice Extracts on Formation of Osteoclasts Induced by RANKL in BMM cells

The experiment was performed in the same manner as Example 6, except that licorice and roasted licorice extracts were used with 5.0 μg/ml or 7.5 μg/ml. As a result, osteoclasts were not observed in BMM cells not treated with RANKL; however, the significantly increasing number of osteoclasts was found in BMM cells treated with RANKL (p <0.0001) (Fig. 12a). The treatment of 5.0 μg/ml of licorice or roasted licorice extracts was shown to decrease the formation of osteoclasts induced by RANKL by 31.3% and 32.0%, respectively; however, in the 7.0 μg/ml-treatment, roasted licorice extracts (51.7%) exhibited much higher inhibition to the formation of osteoclasts than licorice extracts (31.1%) (Fig. 12b, Table 6). TABLE 6

8-2. Comparative Analyses of Inhibitory Effects of Licorice and Roasted Licorice

Extracts on Bone Resorption by Osteoclasts

The experiment was performed in the same manner as Example 7, except that licorice and roasted licorice extracts were used with 5.0 μg/ml or 7.5 μg/ml. The roasted licorice extract at concentration of 7.5 μg/ml was revealed to exert much higher inhibitory effect on the formation of resorption pits than the licorice extract. Accordingly, this result leads us to conclude that extracts of roasted licorice have much higher inhibitory effects on born resorption by osteoclasts than extracts of licorice (Fig. 13).

EXPERIMENTAL EXAMPLE 9: Inhibitory Effect of Roasted Licorice Extracts on Bone Metastasis of Breast Cancer Cells in Animal Model 9-1. Culture of Breast Cancer Cell Line, MDA-MB-231 cells

MDA-MB-231 cells (1 x 10⁵ cells) were placed in a 75T flask and then cultured in L-15 (Leibovitz's-15) media containing 1% antibiotic-antimycotic solution and 10% FBS. Before 24 hr of the injection of MDA-MB-231 cells into the heart of nude mouse, the media was changed with a fresh media. Immediately before the injection of MDA-MB-231 cells into the heart, cells were incubated with trypsin-EDTA and collected with PBS for use in experiments.

9-2. Injection of MDA-MB-231 Cells into Heart of Nude Mouse

For the experiment, 4 week aged female nude mice (Balb/c- nu/nu, Central Lab. Animal Inc., Seoul, Korea) were used. Before the injection with cells, mice were anesthetized by intraperitoneal administration of anesthetics (ketamine 40 mg/kg and xylazine 16 mg/kg). Afterwards, MDA-MB-231 cells (1 x 10⁶ cells/0.1 ml PBS) were injected into the left ventricle of mice using 26 gauge syringes. 9-3. Administration of Roasted Licorice Extract to Nude Mice Injected with Breast

Cancer Cells and CT Imaging

Following 1-week of the injection of MDA-MB-231 cells into nude mice, extracts of roasted licorice were intraperitoneally administered into nude mice for 28 days (three times a week) with dosages of 1 mg/kg (body weight of nude mouse) and 5 mg/kg (body weight of nude mouse). Thereafter, nude mice were sacrificed and their hind limbs were dissected and scanned with micro-computed tomography (SkyScan-1076, Kartuizersweg, Belgium).

9-4. Results

Nude mice injected with MDA-MB-231 cells were observed to have increased portions of bone resorption, compared with them not injected with MDA-MB-231.

The extract of roasted licorice at a dosage of 1 mg/kg exhibited little or no suppressive effect at a dosage of 1 mg/kg on bone resorption; however, at a dosage of 5 mg/kg, it showed significant suppressive effect on bone resorption by metastasis of MDA-MB-231 (Fig. 14).

As such, the roasted licorice extract of this invention provides a composition comprising a natural medicine with no toxicities for preventing or treating various bone diseases such as bone damages associated with bone metastasis of cancer cells, osteoporosis, osteomalacia, rickets, osteitis fibrosa, aplastic bone diseases and metabolic bone diseases. The present composition may be prepared as either drugs or foods.

As described hereinabove, the present composition prevents the formation of osteoclasts to improve a bone mineral density, particularly effectively inhibiting bone resorption by bone metastasis of cancer cells to exhibit excellent therapeutic and preventive efficacies on bone diseases. Furthermore, the extracts of roasted licorice used as active ingredients in the composition are considered to be a safety ingredient in the senses that they have no toxicity to human and no influence on the viability of bone marrow macrophages.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. A composition for preventing or treating a bone disease, which comprises an extract of a roasted licorice as an active ingredient.

2. The composition according to claim 1, wherein the extract of the roasted licorice is prepared by extracting the roasted licorice with water, methanol, ethanol or their combinations.

3. The composition according to claim 1, wherein the bone disease is bone damage associated with bone metastasis of cancer cells, osteoporosis, osteomalacia, rickets, osteitis fibrosa, aplastic bone disease and metabolic bone disease.

4. The composition according to claim 1, wherein the composition inhibits the expression of COX (cyclooxygenase)-2 and RANKL (receptor activator of nuclear factor-κB ligand) and promotes the expression of OPG (osteoprotegerin) in osteoblasts.

5. The composition according to claim 1, wherein the composition inhibits the formation of osteoclasts.

6. The composition according to claim 1, wherein the composition is a pharmaceutical composition or a food composition.

7. A method for preventing or treating a bone disease, which comprises administering to a subject a composition comprising an extract of a roasted licorice as an active ingredient.

8. The method according to claim 7, wherein the extract of the roasted licorice is prepared by extracting the roasted licorice with water, methanol, ethanol or their combinations.

9. The method according to claim 7, wherein the bone disease is bone damage associated with bone metastasis of cancer cells, osteoporosis, osteomalacia, rickets, osteitis fibrosa, aplastic bone disease and metabolic bone disease.

10. The method according to claim I₁ wherein the composition inhibits the expression of COX (cyclooxygenase)-2 and RANKL (receptor activator of nuclear factor-κB ligand) and promotes the expression of OPG (osteoprotegerin) in osteoblasts.

11. The method according to claim 7, wherein the composition inhibits the formation of osteoclasts.