WO2008007880A1

WO2008007880A1 - A composition comprising an extract of prunus persica (l.) batsch for treating and preventing bone diseases

Info

Publication number: WO2008007880A1
Application number: PCT/KR2007/003318
Authority: WO
Inventors: Won Yoon Chung; Sun Kyoung Lee; Kwang Kyun Park; Jae Kwan Hwang; Sang Kook Lee
Original assignee: Industry-Academic Cooperation Foundation, Yonsei University
Priority date: 2006-07-14
Filing date: 2007-07-09
Publication date: 2008-01-17
Also published as: KR20080006907A; KR100823354B1

Abstract

The crude extract, polar solvent soluble or non-polar solvent soluble extract of Prunus persica (L.) BATSCH of the present invention inhibitedthe formation of osteoclasts in mouse bone marrow macrophages treated with RANKL and the bone resorption by osteoclasts without harmful effect on the viability of mouse bone marrow macrophage cells. Accordingly, it can be used as the therapeutics or health food for treating and preventing bone diseases.

Description

A COMPOSITION COMPRISING AN EXTRACT OF PRUNUS PERSICA (L.) BATSCH FOR TREATING AND PREVENTING

BONE DISEASES

Technical Field

[1] The present invention relates to composition comprising an extract of Prunus persica

(L.) BATSCH for treating and preventing bone diseases.

[2]

Background Art

[3] 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 muscles 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). pp. 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., 58f5I pp.391-393, 2003).

[4] Bone remodeling has been reported to involve two types of cells. One of them is bone-forming osteoblasts and another is bone-resorbing osteoclasts. Osteoblasts produce RANKL (receptor activator of nuclear factor-kB 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, pp.795-823, 2002; Wagner EF. et al., Cwrr Opin Genet Dev., 11, pp.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. pp.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., 15(Y). pp.72-80, 2005). It takes about 100 days to disrupt old bones and rebuild new bones (Schwarz EM. Et al., Cwrr Opin Orthop., YV, pp. 329-335, 2000). While 100% of calcium content in bone is changed with 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. 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 J., 93Q). pp.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 microarchitecture that becomes susceptible to skeletal fractures (Stephan JJ. Et al., Endocr Regul., 37(4^*). pp.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, cy- closporine and GnRH), alcohol, smoking or accidents (Rosen CJ., Η Engl J Med., 353(6^*). pp.595-603, 2005 ; Davidson M., Clinicain Reviews., 12(4^*). pp.75-82, 2002).

[5] Breast cancer, prostate cancer and multiple myeloma are accompanied with bone metastasis (Kozlow W. et al., J Mannary Gland Biol Neoplasia., 10(2), pp. 169-180, 2005). The life span of patients having such cancer has been suggested to be dependent on bone metastasis. The bone metastasis observed in breast cancer cells do not affect directly bone but stimulates osteoclasts (Boyde A. et al., Scan Electron Microsc, 4, pp.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. 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 RelatRes., 415S. pp. S19-S31, 2003).

[6] Prunus persica (L.) BATSCH is originated from drainage area of Yellow River in

China, grown in the Korea, America, Italy and Spain. It has been known that the peel, leaf, flower, fruit, seed of downy peach together with peach worm has been used as an oriental medicine since long years ago. Persicae semen has been reported to comprise glyceride, sterol, emulsin and amygdalin and to show inhibitory effect on Oketsu (Kosuge T., Chem. Pharm. Bull, 33, pp.1496-1498, 1985), to be closely involved in anti-tumor promoter and thereby is used as a therapeutic for treating women's diseases (Fukuda, T., Biol. Pharm. Bull, 26, pp.271-273, 2003).

[7] Prunus persica (L.) BATSCH of the present invention is generally classified into white peach and yellow peach. The present inventors investigated the inhibitory effect of the extract or purified extract of white peach and yellow peach on the osteolysis of osteoclast induced by RANKL, and finally found that the crude extract, polar solvent soluble or non-polar solvent soluble extract of Prunus persica (L.) BATSCH shows potent inhibitory effect on the formation of osteoclast induced by RANKL as well as on the bone resorption by osteoclast without harmful effect on the cell viability of macrophage cells derived from mouse bone marrow therefore it can be used as the effective and safe therapeutics or health food for treating and preventing bone disease.

[8]

Disclosure of Invention Technical Problem

[9] The present invention provides a pharmaceutical composition comprising the crude extract, polar solvent soluble or non-polar solvent soluble extract of Prunus persica (L.) BATSCH for the treatment and prevention of bone disease as an active ingredient, together with a pharmaceutically acceptable carrier.

[10] The present invention also provides a use of the crude extract, polar solvent soluble or non-polar solvent soluble extract of Prunus persica (L.) BATSCH for the preparation of therapeutic agent for treating or preventing bone diseases in a mammal including human in need thereof.

[11] The present invention also provides a health food comprising the crude extract, polar solvent soluble or non-polar solvent soluble extract of Prunus persica (L.) BATSCH as an active ingredient for the alleviation and prevention of bone diseases.

[12]

Technical Solution

[13] Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising the crude extract, polar solvent soluble or non-polar solvent soluble extract of Prunus persica (L.) BATSCH for treating or preventing bone diseases as an effective ingredient, together with a pharmaceutically acceptable carrier.

[14] In accordance with another aspect of the present invention, there is also provided a use of the crude extract, polar solvent soluble or non-polar solvent soluble extract of Prunus persica (L.) BATSCH for the preparation of therapeutic agent for treating or preventing bone diseases in a mammal including human in need thereof.

[15] In accordance with the other aspect of the present invention, there is also provided a method for treating or preventing bone diseases comprising administering to mammal in an effective amount of the crude extract, polar solvent soluble or non-polar solvent soluble extract of Prunus persica (L.) BATSCH as an effective ingredient, together with a pharmaceutically acceptable carrier thereof.

[16] The term "crude extract" disclosed herein comprise the extract which can be prepared by extracting plant material with at least one solvent selected from the group consisting of water, C 1 to C 4 lower alcohol such as methanol, ethanol and the like, ethyl acetate, ether and chloroform, preferably, the mixture of water and ethanol.

[17] The term "polar solvent soluble extract" disclosed herein comprise the extract which can be prepared by fractionating above-described crude extract with polar solvent, for example, water, C 1 to C 4 lower alcohol such as methanol, ethanol and the like, or the mixture of thereof, preferably, butanol.

[18] The term "non-polar solvent soluble extract" disclosed herein comprise the extract which can be prepared by extracting above-described crude extract with non-polar solvent, for example, ethyl acetate, hexane, chloroform or methylene chloride, preferably, ethyl acetate.

[19] The term "bone diseases" disclosed herein includes various bone diseases caused by excessive formation of osteoclast induced by RANKL or excessive bone resorption of osteoclast, for example, osteoporosis, rheumatoid arthritis, osteoarthritis, degenerative arthritis, disk, osteomalacia, rickets or polyostotic fibrous dysplasia, preferably, osteoporosis.

[20] The term "extract of Prunus persica (L.) BATSCH" disclosed herein comprises the extract of the flesh, peel or seed of Prunus persica (L.) BATSCH, preferably, the flesh or the peel of Prunus persica (L.) BATSCH.

[21]

[22] Hereinafter, the present invention is described in detail.

[23]

[24] An inventive extract of Prunus persica (L.) BATSCH can be prepared in detail by following procedures.

[25]

[26] For example, the flesh of Prunus persica (L.) BATSCH is washed with removing the seed, mixed with 1 to 40-fold, preferably, approximately 1 to 30-fold volume of water, C 1 to C 4 lower alcohols such as ethanol, methanol and the like, or the mixture of thereof, preferably, the mixture of water and ethanol with the mixed ratio ranging from about 1: 0.1 to 1: 10, more preferably, 70 to 100% ethanol or methanol in water; the solution is treated at the temperature ranging from 0 to 120°C, preferably, at the room temperature for the period ranging from 1 to 24 hours, preferably, 3 to 10 hours with extraction method by the extraction with hot water, cold water, reflux extraction or ultra-sonication extracton, preferably, reflux extraction and then dried by vacuum freeze-drying to obtain crude extract of Prunus persica (L.) BATSCH.

[27] Additionally, the polar solvent soluble and non-polar solvent soluble extract of present invention can be prepared by following procedure; the crude extract prepared by the above-described step, is suspended in water, and then is mixed with 1 to 100-fold, preferably, 1 to 5-fold volume of non-polar solvent soluble such as hexane, ethyl acetate, chloroform, ether and the like; the non-polar solvent soluble layer is collected to obtain non-polar solvent soluble extract of the present invention and remaining polar solvent soluble layer is collected to obtain polar solvent soluble extract of the present invention which is soluble in water, C to C lower alcohol such as methanol, ethanol, butanol and the like or the mixture thereof; the extract is collected with filtration, concentrated under reduced pressure and dried to obtain an inventive polar solvent soluble extract of Prunus persica (L.) BATSCH.

[28] Also, the above-described procedures may be modified or subjected to further step to fractionate or isolate more potent fractions or compounds by conventional procedure well-known in the art, for example, the procedure disclosed in the literature (Harborne J.B. Phytochemical method; A guide to modern thchniques of plant analysis, 3^r Ed. Pp. 6-7, 1998).

[29] It is an object of the present invention to provide a method of treating or preventing bone disease in mammal or human comprising administering to said mammal or human with an effective amount of an extract of Prunus persica (L.) BATSCH together with a pharmaceutically acceptable carrier thereof.

[30] The inventive composition for treating and preventing bone diseasemay comprises the above-described extract as 0.1 ~ 50 % by weight based on the total weight of the composition.

[31] The inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method well known in the art. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co, Easton PA).

[32] Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.

[33] The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

[34] For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents that are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. For topical administration, the extract of the present invention can be formulated in the form of ointments and creams.

[35] Pharmaceutical formulations containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion).

[36] The composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

[37] The desirable dose of the inventive extract or composition varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging 0.1 to 1000mg/kg, preferably, 1 to 100 mg/kg by weight/day of the inventive extract of the present invention. The dose may be administered in single or divided into several times per day. In terms of composition, the amount of inventive extract should be present between 0.01 to 50% by weight, preferably, 0.5 to 40% by weight based on the total weight of the composition.

[38] The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intra-cerebroventricular injection.

[39] Also, it is another object of the present invention to provide a health food or food additives comprising the crude extract, polar solvent soluble or non-polar solvent soluble extract of Prunus persica (L.) BATSCH, together with a sitologically acceptable additive for the prevention and alleviation of bone diseases. The health food of the present invention comprises the above-described extract as 0.01 to 80 %, preferably, 1 to 50 % by weight based on the total weight of the composition.

[40] The health food of the present invention can be contained in health food, health beverage etc, and may be used as powder, granule, tablet, chewing tablet, capsule, beverage etc.

[41] The health food of the present invention comprises the above-described extract as

0.01 to 80 %, preferably, 1 to 50 % by weight based on the total weight of the composition.

[42] The food additive of the present invention can be contained in health food, health beverage etc, and may be used as powder, granule, tablet, chewing tablet, capsule, beverage etc.

[43] Also, the present invention provide a composition of the health food beverage for the prevention and improvement of bone diseases adding 0.01 to 80 % the above-described extract by weight, 0.001 to 5 % amino acids by weight, 0.001 to 2 % vitamins by weight, 0.001 to 20 % sugars by weight, 0.001 to 10 % organic acids by weight and proper amount of sweetener and flavors.

[44] To develop for health food, examples of addable food comprising the above- described extract of the present invention are various food, beverage, gum, vitamin complex, health improving food and the like, and can be used as power, granule, tablet, chewing tablet, capsule or beverage etc.

[45] Also, the extract of the present invention will be able to prevent and alleviate bone disease by way of adding to child and infant food, such as modified milk powder, modified milk powder for growth period, modified food for growth period.

[46] The above-described composition therein can be added to food, additive or beverage, wherein, the amount of above described extract in food or beverage may generally range from about 0.1 to 80w/w %, preferably, 1 to 50 w/w % of total weight of food for the health food composition and 1 to 30 g, preferably, 3 to 10 g on the ratio of IOOD of the health beverage composition.

[47] Providing that the health beverage composition of present invention contains above described extract as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cy- clodextrin; and sugar alcohol such as xylitol, and erythritol etc. As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably, 5 to 12 g in the ratio of 100 D of present beverage composition.

[48] The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition. Examples of addable food comprising aforementioned extract therein are various food, beverage, gum, vitamin complex, health improving food and the like.

[49] The inventive composition may additionally comprise one or more than one of organic acid, such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid; phosphate, such as phosphate, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate; natural anti-oxidants, such as polyphenol, catechin, α- tocopherol, rosemary extract, vitamin C, green tea extract, licorice root extract, chitosan, tannic acid, phytic acid etc.

[50]

Advantageous Effects

[51] The crude extract, polar solvent soluble or non-polar solvent soluble extract of

Prunus persica (L.) BATSCH of the present invention inhibited the formation of osteoclasts in mouse bone marrow macrophages treated with RANKL and the bone resorption by osteoclasts without harmful effect on the viability of mouse bone marrow macrophage cells. Accordingly, it can be used as the therapeutics or health food for treating and preventing bone diseases.

[52]

Brief Description of the Drawings

[53] The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

[54]

[55] Fig. 1 shows the effect of the ethanol extract of white peach and the purified fractions thereof on the cell viability of bone marrow macrophage cell derived from mice; [56] [57] Fig. 2 shows the effect of the ethanol extract of yellow peach and the purified fractions thereof on the cell viability of bone marrow macrophage cell derived from mice; [58] [59] Fig. 3 shows the inhibitory effect of the ethanol extract of flesh part of white peach and the purified fractions thereof on the formation of osteoclast; [60] [61] Fig. 4 shows the inhibitory effect of the ethanol extract of peel part of white peach and the purified fractions thereof on the formation of osteoclast; [62] [63] Fig. 5 shows the inhibitory effect of the ethanol extract of flesh part of yellow peach and the purified fractions thereof on the formation of osteoclast; [64] [65] Fig. 6 shows the inhibitory effect of the ethanol extract of peel part of yellow peach and the purified fractions thereof on formation of osteoclast; [66] [67] Fig. 7 shows the inhibitory effect of the ethanol extract of peel part of white peach and the butanol soluble fraction thereof on the bone resorption by osteoclast; [68] [69] Fig. 8 shows the inhibitory effect of the ethyl acetate soluble fractions of flesh part and peel part of yellow peach on the bone resorption by osteoclast. [70]

Best Mode for Carrying Out the Invention [71] It will be apparent to those skilledin the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention. [72]

Mode for the Invention [73] The present invention is more specifically explained by the following examples.

However, it should be understood that the present invention is not limited to these examples in any manner. [74]

[75] Example 1. Preparation of the ethanol extract of Prunus persica (L.) BATSCH

[76] White peach and yellow peach of the present invention was purchased from

Kyeonggi Dong-boo Fruit Agricultural Cooperative located at Icheon in Korea. [77] The peach was washed with removing the seed, separated into flesh and peel and dried with lyophilization. The flesh part and peel part of peach was mixed with 80% ethanol with the ratio of 1:5 (w/v) and the mixture was subjected to reflux extraction for 48 hours, 3 times. The solution was filtered with filter paper No.2 (Advantec TOYO, Japan) and the filtrate was collected. The collected residue was evaporated by rotary vacuum evaporator (Heidolph VV2011, Switzerland), dried with lyophilization to obtain various kinds of ethanol extract of peach, i.e., 14.05g of the peel extract of white peach, 13.6Og of the peel extract of white peach, 13.96g of the flesh extract of yellow peach, 13.75g of the peel extract of yellow peach respectively, to use as test samples in following experiments.

[78]

[79] Example 2. Preparation of the polar solvent and non-polar solvent extract of

Primus pcrsica (L.) BATSCH

[80] The ethanol extract prepared in Example 1 was suspended in water and the suspension was fractionated with ethyl acetate, butanol and distilled water to afford ethyl acetate-soluble fraction, butanol-soluble fraction and water-soluble fraction of the present invention. The fractions were filtered and the filtrates were evaporated to obtain ethyl acetate-soluble fraction as a non-polar solvent soluble extract and the butanol-soluble fraction and water-soluble fraction as polar solvent soluble extract of the present invention.

[81] All the fractions prepared from the above-described method were stored at -20°C to use as test samples.

[82]

[83] Experimental Example 1. Cell toxicity test of white peach extract

[84] In order to examine the cell toxicity test using by the ethanol extract ofPrunus persica (L.) BATSCH and the fraction thereof prepared in Example 1 and 2, following experiment was performed according to the method disclosed in the literature (Wattel A. et al., J. Cell Biochem., VI(IY pp.285-295, 2004).

[85] Mouse bone marrow macrophage cell (1 x 10⁴ cells) was added to each well of 96 well plates and cultured in α-MEM containing 1% of antibiotic-antimycotic solution, 10% of FBS (fetal bovine serum), 30 ng/ml of M-CSF (macrophage-colony stimulating factor), and the ethanol extract of flesh part of white peach, the ethanol extract of peel part of white peach, the ethyl acetate soluble fractions thereof, the butanol soluble fractions thereof, or the water soluble fractions thereof as test samples. The test samples prepared in Example 1 and 2 were dissolved in DMSO (dimethyl sulfoxide) and water, and the solutions were diluted with 10% FBS-α-MEM to the concentration of 0, 1, 10, 100 D/ml. The mouse bone marrow macrophage cells were cultured by changing with fresh new medium every 2 days, and incubated for 5 days at 37 °C in 5% CO incubator. MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was added thereto to the concentration of 5 mg/ml per each well. After culturing for 4 hours at 37 °C, the medium and MTT solution were removed and then 200 D of DMSO per each well was added thereto. 20 minutes after the reaction, the absorbance was measured at 570nm. The cell viability was calculated by the percentage ratio of absorbance of test sample groups comprising the extract ofPrunus persica (L.) BATSCH or the fraction thereof to the absorbance of control treated with only medium.

[86] As can be seen in Tables 1, 2 and Fig. 1, the cell viability of the groups treated with the ethyl acetate-soluble fraction and the butanol-soluble fraction of flesh part of white peach; and the ethyl acetate-soluble fraction of peel part of white peach in the concentration of 100D/ml were reduced by 21%, 13% and 61% respectively (IC of the

50 ethyl acetate-soluble fraction of peel part of white peach = 90.24 D/ml). However, the ethanol extract and butanol-soluble fraction and water soluble fraction of flesh part of white peach; and the butanol-soluble fraction and water-soluble fraction of peel part of white peach in the concentration of 0-100 D/ml did not show any effect on the cell viability of the mouse bone marrow macrophage cell. [87] Table 1

[Table 1] [Table ]

[88] Table 2

[Table 2] [Table ]

[89] [90] Experimental Example 2. Cell toxicity test of the extract of yellow peach [91] In order to examine the cell toxicity test using by the ethanol extract of yellow peach and the fractions thereof prepared in Example 1 and 2, following experiment was performed according to procedure similar to procedure disclosed in the Experimental Example 1.

[92] As can be seen in Tables 3, 4 and Fig. 2, the cell viability of the groups treated with the ethyl acetate-soluble fraction of flesh part and peel part of yellow peach were reduced by 25%, and 27% respectively. However, the ethanol extract and butanol- soluble fraction and water soluble fraction of flesh part and peel part of yellow peach in the concentration of 0-100 D/ml did not show any effect on the cell viability of the mouse bone marrow macrophage cell.

[93] Table 3 [Table 3] [Table ]

[94] Table 4

[Table 4] [Table ]

[95] [96] Experimental Example 3. The inhibitory effect of the extract of white peach on the formation of osteoclasts

[97] In order to examine the inhibitory effect of the extract and the fractions of white peach prepared in Example 1 and 2 on the formation of osteoclasts isolated from mouse bone marrow macrophage, following experiments were performed according to the method disclosed in the literature (Park EK. et al., Biochem Biophys Res Commun., 325f4I pp.1472- 1480, 2004).

[98] Mouse bone marrow macrophage cells were added to 96 well plates in a concentration of IxIO⁴ cells per each well and incubated in 10% FBS-α-MEM containing RANKL (100 ng/ml), M-CSF (30 ng/ml), and 100 D/ml of the ethanol extract of flesh part or peel part of white peach or 100 D/ml of ethyl acetate-soluble fraction, butanol- soluble fraction, or water-soluble fraction of flesh part or peel part of white peach. 75, 100 D/ml of ethanol extract of peel part of white peach and 75, 100 D/ml of ethyl acetate- soluble fraction, butanol-soluble fraction or water-soluble fraction of peel part of white peach were incubated in 10% FBS-α-MEM. The group treated with only 10% FBS- α-MEM was used as control. The cells were cultured with changing fresh medium every 2 days for 5 days and then the formation of osteoclasts was examined by staining with TRAP analysis kit (tartrate-resistant acid phosphatase assay kit; Sigma, MO, USA). The number of cells containing more than three nuclei was counted by using optical microscope.

[99] As can be seen in Table 5 and Fig. 3, the number of osteoclasts treated with RANKL were significantly increased whereas the mouse bone marrow macrophage cells not- treated with RANKL did not form osteoclast cells (p<0.00001). The mouse bone marrow macrophage cells treated with the ethanol extract of flesh part of white peach and various fractions of flesh part of white peach inhibited the formation of RANKL- induced osteoclasts by 24.5% (p<0.05). The mouse bone marrow macrophage cells treated with the ethanol extract of peel part of white peach and various fractions of peel part of white peach inhibited the formation of RANKL-induced osteoclasts in a dose dependent manner (ethanol extract: p<0.05, butanol-soluble fraction: p<0.001) as can be seen in Table 6 and Fig. 4. It is confirmed that the ethyl acetate-soluble fraction of peel part of white peach completely inhibit the formation of osteoclast and such result is assumed that the fraction significantly reduced the cell viability of mouse bone marrow macrophage cells resulting in blocking of osteoclast differentiation.

[100] Table 5 [Table 5] [Table ]

[101] Table 6 [Table 6] [Table ]

[102]

[103] Experimental Example 4. The inhibitory effect of yellow peach extract on the formation of osteoclasts

[104] In order to examine the inhibitory effect of the extract and fractions of yellow peach prepared in Example 1 and 2 on the formation of osteoclasts isolated from mouse bone marrow macrophage, following experiments were performed according to the method similar to the procedure disclosed in the Experimental Example 3.

[105] 100 D/ml of the ethanol extract, ethyl acetate-soluble fraction, butanol-soluble fraction or water-soluble fraction of flesh part of yellow peach were treated to mouse bone marrow macrophage cells respectively and 75, 100 D/ml of the ethanol extract and 75, 100 D/ml of ethyl acetate-soluble fraction, butanol-soluble fraction or water-soluble fraction of peel part of yellow peach were treated in 10% FBS-α-MEM to incubate for 5 days. The number of osteoclasts was determined.

[106] As can be seen in Tables 7, 8 and Figs. 5 and 6, the number of osteoclasts treated with RANKL were significantly increased whereas the mouse bone marrow macrophage cells not-treated with RANKL did not form osteoclast cell(p<0.00001). the ethanol extract and various fractions of flesh part and peel part of yellow peach significantly decreased the formation of osteoclasts (p<0.0001).

[107] Table 7 [Table 7] [Table ]

[108] Table 8 [Table 8] [Table ]

[109] [HO] Experimental Example 5. Determination of bone resorption by osteoclasts [111] In order to examine the effect of the extract and fractions of yellow peach prepared in Example 1 and 2 on the bone resorption induced by RANKL in mouse bone marrow macrophage cells, following experiments were performed according to the method disclosed in the literature (Wattel A. et al., J. Cell Biochem., 92f2^*>. pp.285-295, 2004).

[112] The ethanol extract and butanol-soluble fraction of peel part of white peach and ethyl acetate-soluble fraction of flesh part and peel part of yellow peach showing potent inhibiting activity of the formation of osteoclasts induced by RANKL were adopted as test samples in the experiment.

[113] The mouse bone marrow macrophage cells separated from the hind legs of 4- weeks aged mice were added to 24 well plates coated with calcium phosphate with a concentration of 5xlO⁴ cells per each well. 10% FBS-α-MEM containing RANKL (100 ng/ml), M-CSF (30 ng/ml), the ethanol extract of peel part of white peach (75, 100 D/ml) and butanol-soluble fraction of peel part of white peach (75, 100 D/ml) was added to each well. The ethyl acetate-soluble fraction of flesh part of yellow peach (10, 50 D/ml) and the ethyl acetate-soluble fraction of peel part of yellow peach (10, 50 D/ml) were added thereto to incubate for 15 days with changing fresh medium every 2 days. After the incubation, the medium was removed and sodium hypochlorite solution was added thereto. 5 minutes after the reaction, the sodium hypochlorite solution was removed and plate was washed with distilled water twice. The resorption pit formed by osteoclasts was observed by optical microscope.

[114] As can be seen in Fig. 7 the ethanol extract of peel part of white peach and the butanol-soluble fraction of peel part of white peach significantly inhibited the formation of resorption pit in a does dependent manner.

[115] As can be seen in Fig. 8, the ethyl acetate-soluble fractions of flesh part and peel part of yellow peach significantly inhibited the formation of resorption pit in does dependent manner.

[116]

[117]

[118] Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

[119]

[120]

[121] Preparation of powder

[ 122] Extract of Prunus persica (L.) BATSCH of Example 1 20mg

[123] Lactose lOOmg

[124] Talc lOmg

[125] Powder preparation was prepared by mixing above components and filling sealed package.

[126]

[127] Preparation of tablet

[ 128] Extract of Prunus persica (L.) BATSCH of Example 1 1 Omg

[ 129] Corn Starch 1 OOmg [130] Lactose lOOmg

[131] Magnesium Stearate 2mg

[132] Tablet preparation was prepared by mixing above components and entabletting.

[133]

[134] Preparation of capsule

[135] Extract of Prunus persica (L.) BATSCH of Example 1 1 Omg

[136] Corn Starch 3mg

[137] Lactose 14.8mg

[138] Magnesium Stearate 0.2mg

[139] Capsule preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method. [140]

[141] Preparation of injection

[142] Extract of Prunus persica (L.) BATSCH of Example 1 lOmg

[143] Mannitol 180mg

[ 144] Distilled water for inj ection 2974mg

[145] Na₂HPO₄, 12H₂O 26mg

[146] Injection preparation was prepared by dissolving the components in 2D ample and sterilizing by conventional injection preparation method. [147]

[148] Preparation of health care food

[149] Extract of Prunus persica (L.) BATSCH of Example 1 lOOOmg

[150] Vitamin mixture optimum amount

[151] Vitamin A acetate 70mg

[152] Vitamin E l. Omg

[153] Vitamin B₁ 0.13mg

[154] Vitamin B 0.15mg

[155] Vitamin B6 0.5mg

[156] Vitamin B 12 0.2mg

[157] Vitamin C l Omg

[158] Biotin lOmg

[159] Amide nicotinic acid 1.7mg

[160] Folic acid 50mg

[161] Calcium pantothenic acid 0.5mg

[162] Mineral mixture optimum amount

[163] Ferrous sulfate 1.75mg

[ 164] Zinc oxide 0.82mg

[165] Magnesium carbonate 25.3mg [166] Monopotassium phosphate 15mg

[167] Dicalcium phosphate 55mg

[168] Potassium citrate 90mg

[169] Calcium carbonate lOOmg

[170] Magnesium chloride 24.8mg

[171] The above mentioned vitamin and mineral mixture may be varied in may ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.

[172]

[173] Preparation of health beverage

[ 174] Extract of Prunus persica (L.) BATSCH of Example 1 1 OOOmg

[175] Citric acid lOOOmg

[176] Oligosaccharide lOOg

[177] Apricot concentration 2g

[178] Taurine Ig

[179] Concentrated plum solution 2g

[ 180] Distilled water 900D

[181] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85⁰C for 1 hour, filtered and then filling all the components in 2000D ample and sterilizing by conventional health beverage preparation method.

[182]

[183] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

[184]

Industrial Applicability

[185] As described in the above, the crude extract, polar solvent soluble or non-polar solvent soluble extract of Prunus persica (L.) BATSCH of the present invention inhibited the formation of osteoclasts in mouse bone marrow macrophages treated with RANKL and the bone resorption by osteoclasts without harmful effect on the viability of mouse bone marrow macrophage cells. Accordingly, it can be used as the therapeutics or health food for treating and preventing bone diseases.

Claims

[1] A pharmaceutical composition comprising the crude extract, polar solvent soluble or non-polar solvent soluble extract of Prunus persica (L.) BATSCH for treating or preventing bone diseases as an effective ingredient, together with a pharmaceutically acceptable carrier.

[2] The pharmaceutical composition of claim 1, wherein said extract is extracted from the flesh part or peel part of Prunus persica (L.) BATSCH.

[3] The pharmaceutical composition of claim 1, wherein said crude extract is prepared by extracting plant material with at least one solvent selected from the group consisting of water, C 1 to C 4 lower alcohol such as methanol, ethanol and the like, ethyl acetate, ether and chloroform.

[4] The pharmaceutical composition of claim 1, wherein said polar solvent soluble extract is prepared by fractionating said crude extract with water or C to C lower alcohol.

[5] The pharmaceutical composition of claim 1, wherein said non-polar solvent soluble extract is prepared by fractionating said crude extract with ethyl acetate, hexane, chloroform or methylene chloride.

[6] The pharmaceutical composition of claim 1, wherein said bone disease is osteoporosis, rheumatoid arthritis, osteoarthritis, degenerative arthritis, disk, osteomalacia, rickets or polyostotic fibrous dysplasia.

[7] A use of the crude extract, polar solvent soluble or non-polar solvent soluble extract of Prunus persica (L.) BATSCH for the preparation of therapeutic agent for treating or preventing bone diseases in a mammal including human in need thereof.

[8] A method for treating or preventing bone diseases comprising administering to mammal in an effective amount of the crude extract, polar solvent soluble or non-polar solvent soluble extract of Prunus persica (L.) BATSCH as an effective ingredient, together with a pharmaceutically acceptable carrier thereof.

[9] A health care food comprising the crude extract, polar solvent soluble or non- polar solvent soluble extract of Prunus persica (L.) BATSCH, together with a sitologically acceptable additive for prevention and alleviation of bone diseases.

[10] The health care food according to claim 9 wherein said health care food is provided as powder, granule, tablet, capsule or beverage type.