KR101256925B1 - Composition for Preventing or Treating Bone Disease - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating a bone disease comprising a compound of a specific structure.
The composition of the present invention exhibits great efficacy in preventing and treating bone diseases by inhibiting the formation of osteoclasts and is safe because it is derived from natural sources.

Description

Composition for Preventing or Treating Bone Disease

The present invention relates to a composition for preventing or treating bone diseases.

More specifically, the present invention relates to a pharmaceutical composition and a food composition for the prevention or treatment of bone diseases comprising a specific compound isolated from peach as an active ingredient.

The bone supports the body's soft tissues and body weight and surrounds the internal organs to protect the internal organs from external impact. It is also an important part of the human body that not only structurally supports muscles or organs but also stores calcium and other essential minerals in the body, such as phosphorus and magnesium. Thus, the bones of grown adults do not stop, and until the day they die, they maintain a very dynamic and continuous regeneration of the process of producing and absorbing the old bones and replacing them with new bones. This is called bone remodeling (Yamaguchi A. et al., Tanpakushitsu Kakusan Koso . , 50 (6 Suppl): 664-669 (2005)). Bone turnover, which removes old bone and replaces it with new bone, is essential for restoring bone damage caused by growth and stress and maintaining proper bone function (Cohen-Solal M. et al., Therapie . , 58 (5): 391-393 (2003).

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

Osteoporosis is a condition in which bone mass decreases due to various causes and the risk of fracture is continuously increased due to the deterioration of the microstructure of bone tissue. Osteoporosis is a condition in which minerals (particularly calcium) and substrates that make up bone are reduced. Formation is broken, resulting in osteoclasts being increased than osteogenic (Iqbal MM., South Med J. , 93 (1): 2-18 (2000)). Normal bone inside has a dense structure like a net, but in the case of osteoporosis, the gap between the structures becomes wider and the microstructure becomes thinner and weaker, so that the bone can be easily fractured even with a small impact (Stepan JJ. Et al. , Endocr Regul. , 37 (4): 225-238 (2003). Osteoporosis occurs rapidly in postmenopausal postmenopausal osteoporosis, with rapid bone loss (2% to 3% per year) at the beginning of menopause, increasing the risk of compression of the spine and fracture of the carpal bone. Senile osteoporosis, which causes progressive bone loss in the hip and vertebrae (0.5-1% per year), and diseases (endocrine diseases, gastrointestinal diseases, malignancies) or drugs of any age. (adrenocortical hormone, anticancer chemotherapy, thyroid hormone, anticonvulsants, anticoagulants, methotexate, cyclosporine, GnRH, etc.), alcohol, smoking, and are classified as secondary osteoporosis (secondary osteoporosis) caused by accidents (Rosen CJ., N Engl J Med . , 353 (6): 595-603 (2005); Davidson M., Clinicain Reviews . , 12 (4): 75-82 (2002)).

Bone metastasis almost always occurs in patients with breast cancer, prostate cancer or multiple myeloma (Kozlow W. et al., J Mammary Gland Biol Neoplasia . , 10 (2): 169-180 (2005)), and how long these cancer patients can live is known to be affected by bone metastasis. The reason why the mortality rate increases in breast cancer patients and prostate cancer patients is because cancer cells selectively metastasize to bone. Most bone metastases observed in breast cancer are osteolytic metastasis that destroy bone and are known to occur when breast cancer cells stimulate osteoclasts rather than directly affect bone (Boyde A. et al., S can Electron Microsc. , 4: 1537-1554). On the other hand, bone metastases observed in prostate cancer are osteoblastic metastasis. Osteoblasts are also known to be closely associated with osteolysis. Cancer cells that have metastasized to bone proliferate in the microenvironment around the bone and stimulate osteoclast or osteoblast activity to determine whether to progress to osteolytic bone metastasis or osteoblast metastasis (Choong PF. Et al., Clin Orthop Relat). Res . 415 (S): S 19-S 31 (2003)). About 80% of breast cancer patients develop bone metastasis of cancer cells, and the metastasized breast cancer cells activate osteoclasts (Bendre M., et al., Clin Orthop Relat Res. 415 (Suppl): S39-S45 (2003); Palmqvist P. et al., J Immunol ., 169 (6): 3353-3362 (2002)). Activated osteoclasts disrupt the balance of the microenvironment around bones, leading to osteolysis, which often causes pathological fractures, as well as leukoerythroblastic anaemia, bone malformations, and hypercalcemia. ), Bone-related diseases such as pain, nerve-compression syndromes (Roodman GD., N Engl J Med . , 350: 1655-1664 (2004)).

Peach tree (Prunus persica (L.) Batsch has its origin in the highlands of the Yellow River, China, and is currently cultivated in the United States, Italy and Spain, including Korea, and bark in traditional medicine in China and Korea. , Leaves, flowers, berries, peaches, peaches, peach bugs are all known to be used as drugs. Among them, peach seed (Semen Persicae) is composed of glyceride (steride), sterol (sterol), emulsin (amyulda), amygdalin (main ingredient), and is effective in blocking peripheral blood circulation disorder (Oketsu) Reported (Kosuge T.,Chem . Pharm . Bull, 33: 1496-1498 (1985)), which are closely associated with strong anti-tumor promoters and are known to be used to treat gynecological diseases (Fukuda, T.,Biol . Pharm . Bull, 26: 271-273 (2003).

The peach blossom (Flos Persicae) contains ingredients such as camphorol and coumarin, and it is known that folk remedies are effective in treating diseases such as diarrhea, faeces, species, and each other. Peach leaves are also known to be used for the treatment of anxiety headaches, eczema, swelling, and nasal bleeding. The peach fruit used in this study is also known as Persicae fructus, and has a white flesh of white flesh. Peaches are alkaline foods that boost immunity and appetite in oriental medicine, are good for poor development and night blindness, and soften the intestines, eliminating constipation and releasing blood. It is also said that the bark detoxifies and the organic acid removes nicotine and eliminates toxicity. Its main components are water and sugar, and it contains about 1% of organic acids such as tartaric acid, malic acid, and citric acid. It is also rich in vitamin A, esters such as formic acid, acetic acid, and bareric acid, alcohols, aldehydes, and pectin. The pulp contains many free amino acids, especially aspartic acid. The unique aroma is known to be produced by combining esters, alcohols, and aldehydes.

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

The inventors have sought to develop substances that can effectively treat or prevent bone diseases such as osteoporosis and are safe for humans, in particular natural-derived substances. As a result, specific compounds isolated from peaches significantly reduce the formation of RANKL-induced osteoclasts, which are very effective in treating or preventing bone diseases, and each compound does not affect the cell viability of bone marrow macrophages isolated from mice. By confirming that, this invention was completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone diseases comprising a specific compound isolated from peach as an active ingredient.

Another object of the present invention to provide a food composition for the prevention or improvement of bone diseases comprising a specific compound isolated from peach as an active ingredient.

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

According to one aspect of the invention, the present invention is a compound of formula (1); Pharmaceutically acceptable salts thereof; Or it provides a pharmaceutical composition for the prevention or treatment of bone diseases comprising a solvate or hydrate thereof as an active ingredient.

Formula 1

R ₁ to R ₃₁ in Formula 1 are each independently hydrogen, halo, cyano, amino, nitro, C ₁ -C ₁₂ alkyl, C ₂ -C ₆ alkenyl group, C ₃ -C ₈ cycloalkyl, C _5- C ₇ cycloalkenyl, C ₁ -C ₆ alkylamino; Rx and Ry are each independently hydrogen or straight or branched substituted or unsubstituted C _1-12 alkyl.

According to another aspect of the invention, the present invention provides a food composition for the prevention or improvement of bone diseases comprising the compound of Formula 1, its salts or solvates or hydrates thereof as an active ingredient.

The inventors have sought to develop substances that can effectively treat or prevent bone diseases such as osteoporosis and are safe for humans, in particular natural-derived substances. As a result, the compound of formula 1 isolated from peach significantly reduces the formation of RANKL-induced osteoclasts, and is very effective for treating or preventing bone diseases, and each compound affects the cell survival rate of bone marrow macrophages isolated from mice. The present invention has been completed by confirming that the present invention is not affected.

As used herein, the term “halo” as used to define a compound of Formula 1 refers to a halogen group element, including, for example, fluoro, chloro, bromo and iodo.

The term "alkyl" refers to a straight chain or branched, unsubstituted or substituted saturated hydrocarbon group, and includes, for example, methyl, ethyl, propyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and the like. C ₁ -C ₁₂ alkyl means an alkyl group having an alkyl unit having 1 to 12 carbon atoms, and when C ₁ -C ₁₂ alkyl is substituted, the carbon number of the substituent is not included.

The term “cycloalkyl” refers to a cyclic hydrocarbon radical, which includes cyclopropyl, cyclobutyl and cyclopentyl. C ₃ -C ₈ cycloalkyl means a cycloalkyl having 3-8 carbon atoms to form a ring structure, and C ₃ -C ₈ When cycloalkyl is substituted, the carbon number of the substituent is not included.

The term “alkenyl” refers to a straight or branched unsubstituted or substituted unsaturated hydrocarbon group having the specified carbon number, for example ethenyl, vinyl, propenyl, allyl, isopropenyl, butenyl, isobutenyl, t -butenyl , n -pentenyl and n -hexenyl. C ₂ -C ₆ Alkenyl means an alkenyl group having an alkenyl unit having 1 to 6 carbon atoms, and when C ₂ -C ₆ alkenyl is substituted, carbon atoms of the substituent are not included.

The term “cycloalkenyl” refers to a cyclic hydrocarbon group having at least one double bond, such as cyclopentene, cyclohexene and cyclohexadiene. C ₃ -C ₈ cycloalkenyl means cycloalkenyl having 3-8 carbon atoms to form a ring structure, and C ₃ -C ₈ When cycloalkenyl is substituted, the carbon number of the substituent is not included.

The term "pharmaceutically acceptable salt" means a formulation of a compound that does not cause severe irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. The pharmaceutical salt is a compound of the present invention, such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, inorganic acids such as phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and the like, sulfonic acid, tartaric acid, formic acid, citric acid, acetic acid, trichloro It can be obtained by reaction with organic carboxylic acids such as roacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid and the like. In addition, the compound of the present invention is reacted with a base, and salts such as alkali metal salts such as ammonium salts, sodium or potassium salts, and alkaline earth metal salts such as calcium or magnesium salts, dicyclohexylamine, and N-methyl-D-glu It may be obtained by forming salts of organic bases such as carmine and tris (hydroxymethyl) methylamine, and amino acid salts such as arginine and lysine.

The term "hydrate" includes a compound of the present invention comprising a stoichiometric or non-stoichiometric amount of water bound by a non-covalent intermolecular force. Or salts thereof.

The term "solvate" refers to a compound of the present invention or a salt thereof comprising a stoichiometric or nonstoichiometric amount of solvent bound by noncovalent intermolecular forces. Preferred solvents therein are volatile, nontoxic, and / or solvents suitable for administration to humans.

In a preferred embodiment, R ₁ of Formula ₁ To R ₃₁ are each independently hydrogen or alkyl of C ₁ -C ₅ , and each of Rx or Ry is independently hydrogen or alkyl of C ₁ -C ₅ , more preferably R ₁ To R ₃₁ are each independently hydrogen or methyl, and each of Rx or Ry is independently hydrogen or methyl.

In a still more preferred embodiment, the compound of Formula 1 is represented by the following formula (2) or formula (3).

(2)

(3)

The compounds of the present invention may have one or more chiral centers and / or geometric isomeric centers, and thus the present invention is directed to all stereoisomers represented by any one of Formulas 1 to 3, ie, optical isomers, diastereomers and Geometric isomers.

The compounds used as an active ingredient in the composition of the present invention may be separated from crude extracts of flesh (pulp) and / or skin (fruit) of white peach fruit, wherein the peach crude extract is water, alcohol or its The combination may be obtained by adding peach.

Crude extracts as defined herein include water, including purified water, lower alcohols having 1 to 4 carbon atoms or extracts soluble in one or more solvents selected from the group consisting of propylene glycol, ethyl acetate, butylene glycol, ether, chloroform.

The polar solvent soluble extract as defined herein is an extract soluble in water, methanol, butanol or a mixed solvent thereof, preferably butanol, and the non-polar solvent soluble extract is selected from hexane, chloroform, methylene chloride and ethyl acetate. It means the extract available in the same non-polar solvent.

The compounds used as an active ingredient in the composition of the present invention inhibits the formation of osteoclasts, preferably characterized by inhibiting the formation of osteoclasts induced by the receptor activator of NFκB ligand (RANKL). Therefore, it is very effective for preventing or treating bone diseases.

The bone diseases that the composition of the present invention can be prevented or treated include, for example, osteoporosis, osteomalacia, rickets disease, bone metastasis of cancer cells, bone damage caused by bone metastasis of cancer cells, bone lysis due to bone metastasis of cancer cells, fibrous dysplasia , Aplastic bone disease, metabolic bone disease, rheumatoid arthritis, osteoarthritis, degenerative arthritis, and discs, but are not necessarily limited thereto.

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

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The daily dosage of the pharmaceutical composition of the present invention is, for example, 0.001-100 mg / kg.

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

When the composition of the present invention is provided with a food composition, it includes ingredients which are commonly added in the preparation of food, for example, flavoring agents or natural carbohydrates may be included as additional ingredients. For example, natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrins, cyclodextrins, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, and the like). As the flavoring agent, natural flavoring agents (e.g., taumartin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used.

According to another aspect of the present invention, the present invention provides a functional food for preventing or improving bone diseases comprising the food composition.

Functional food means a food whose pharmacological effect is recognized through individual certification by the Food and Drug Administration.

Functional food of the present invention is a food prepared by adding the compound 1, the active ingredient of the present invention to food materials, such as beverages, teas, spices, gums, confectionery, or the like, encapsulated, powdered, suspension, etc. Means bringing a specific effect on health, but unlike the general medicine has the advantage that there are no side effects that can occur when taking a long-term use of the drug as a raw material.

In the functional food of the present invention, the amount of the compound represented by Chemical Formula 1 may not be uniformly defined depending on the type of food, but may be added within a range that does not impair the original taste of the food. In the form of tablets, capsules or beverages, the amount of the food may be added in a range of usually 0.01 to 100% by weight, preferably 1 to 100% by weight, based on the food.

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

(Iii) The present invention provides a composition for preventing, ameliorating or treating a bone disease comprising a compound of a specific structure.

(Ii) The composition of the present invention exhibits great efficacy in preventing and treating bone diseases by inhibiting the formation of osteoclasts and is safe because it is derived from a natural source.

Figure 1 shows the ₁ H-NMR data of 2α, 3α-dihydroxyolean-12-en-28-oic acid isolated from the peach peel.
Figures 2a and 2b shows the ¹³ C-NMR data of 2α, 3α-dihydroxyolean-12-en-28-oic acid isolated from peach peel.
3 is a diagram showing the mass spectrometry data of 2α, 3α-dihydroxyolean-12-en-28-oic acid isolated from peach peel.
Figure 4 is a diagram showing the ¹ H-NMR data of 2α, 3α-dihydroxyurs-12-en-28-oic acid isolated from the peach peel.
5 is a diagram showing ¹³ C-NMR data of 2α, 3α-dihydroxyurs-12-en-28-oic acid isolated from peach skin.
6 is a diagram showing the mass spectrometry data of 2α, 3α-dihydroxyurs-12-en-28-oic acid isolated from peach peel.
7 is a mouse bone marrow versus olenolic acid and ursolic acid as a positive control with 2α, 3α-dihydroxyolean-12-en-28-oic acid, 2α, 3α-dihydroxyurs-12-en-28-oic acid isolated from peach skin A diagram showing data of cell viability for phagocytes.
FIG. 8 is a positive control group of 2α, 3α-dihydroxyolean-12-en-28-oic acid, 2α, 3α-dihydroxyurs-12-en-28-oic acid, isolated from peach skin and induced by RANKL of olenolic acid and ursolic acid. The data shows the inhibitory effect on the formation of osteoclasts.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

Example

Isolation and Structural Identification of Active Ingredients in Peaches

Baekdo used in the present invention was purchased from Gyeonggi-do Dongbu Fruit and Agricultural Cooperatives. After washing the peaches, the seeds were removed, separated into pulp and rind and lyophilized. 80% methanol was added to the skin at a ratio of 1: 5 (w / v) and mixed, and the mixture was extracted three times under reflux for 48 hours at room temperature. The methanol extract was dissolved in an aqueous solution, extracted with ethyl acetate, and concentrated under reduced pressure with a rotary vacuum evaporator (Heidolph VV2011, Switzerland) to obtain an ethyl acetate fraction (1.5 g). Ten fractions of ethyl acetate were passed from methanol / water (4/1, v / v) through methanol 100% solvent through a column filled with reverse-phase (18) silica gel resin (5.0x50.0 cm). The fraction 3 (200.0 mg) +4 (100.8 mg) was separated into a column (3.0x45 cm) filled with silica gel resin with hexane / ethyl acetate (1/1. V / v) to obtain an active fraction. Preparative HPLC using RP-18 column and methanol / water (95/5, v / v) solvent (Hitachi HPLC system: pump L-6700, detector: L-7400, column: YMC J'Sphere ODS-H80, 250x20) mm id) was performed to obtain compounds PS-1 (3.2 mg) and PS-2 (4.3 mg). PS-1 is an oleanolic acid derivative, 2α, 3α-dihydroxyolean-12-en-28-oic acid of formula (1), PS-2 is a ursolic acid derivative, 2α of formula (2) , 3α-dihydroxyurs-12-en-28-oic acid was identified.

Formula 2: 2α, 3α-dihydroxyolean-12-en-28-oic acid

HR-ESI MS m / z 473.3651 [M + H], C ₃₀ H ₄₈ O ₄ (FIG. 1).

¹ H NMR data (400 MHz, pyridine-d ₅ ) δ 5.48 (1H, t, J = 3.6 Hz, H-12), 4.31 (1H, dt, J = 11.2, 3.4 Hz, H-2), 3.78 ( 1H, d, J = 2.8 Hz, H-3), 3.29 (1H, dd, J = 13.6, 3.8 Hz, H-18), 1.29 (3H, s, H-23), 1.19 (3H, s, H -27), 1.03 (3H, s, H-26), 1.00 (3H, s, H-30), 0.98 (3H, s, H-25), 0.93 (3H, s, H-24), 0.91 ( 3H, s, H-29) (FIG. 2).

¹³ C NMR (75 MHz, pyridine-d ₅ ) δ 180.8 (C-28), 145.3 (C-13), 122.9 (C-12), 79.7 (C-3), 66.5 (C-2), 49.1 ( C-5), 48.4 (C-9), 47.0 (C-19), 46.9 (C-17), 43.1 (C-14), 42.6 (C-1), 42.4 (C-18), 40.4 (C -8), 39.2 (C-4), 39.1 (C-10), 34.6 (C-21), 33.6 (C-7), 33.6 (C-22), 33.6 (C-29), 31.3 (C- 20), 29.9 (C-23), 28.7 (C-15), 26.5 (C-27), 24.3 (C-11), 24.2 (C-30), 24.1 (C-16), 22.7 (C-24 ), 18.9 (C-6), 17.9 (C-26), 17.0 (C-25) (FIG. 3).

Formula 3: 2α, 3α-dihydroxyurs-12-en-28-oic acid

HR-ESI MS m / z 473.3681 [M + H], C ₃₀ H ₄₈ O ₄ (FIG. 4).

¹ H NMR data (400 MHz, pyridine-d ₅ ) δ 5.47 (1H, brs, H-12), 4.31 (brd, J = 14.4, H-2), 3.78 (1H, d, J = 2.4 Hz, H -3), 2.63 (1H, d, J = 14.4 Hz, H-18), 1.29 (3H, s, H-23), 1.13 (3H, s, H-24), 1.06 (3H, s, H- 26), 0.98 (3H, d, J = 6.4 Hz, H-29), 0.97 (3H, s, H-25), 0.94 (3H, d, J = 6.4 Hz, H-30), 0.91 (3H, s, H-24) (FIG. 5).

¹³ C NMR (75 MHz, pyridine-d ₅ ) δ 180.3 (C-28), 139.7 (C-13), 126.0 (C-12), 79.7 (C-3), 66.5 (C-2), 53.9 ( C-18), 49.1 (C-5), 48.4 (C-17), 48.3 (C-9), 43.3 (C-1), 43.0 (C-14), 40.6 (C-8), 39.8 (C -4), 39.8 (C-19), 39.2 (C-20), 39.0 (C-10), 37.8 (C-22), 33.9 (C-7), 31.5 (C-21), 29.8 (C- 23), 29.0 (C-15), 25.3 (C-16), 24.1 (C-27), 24.1 (C-11), 22.7 (C-24), 21.8 (C-30), 18.8 (C-6 ), 17.9 (C-26), 17.1 (C-29), 17.1 (C-25) (FIG. 6).

Cell culture

Four-week-old male ICR mice (Central Experimental Animal Co., Ltd., Seoul, South Korea) were removed from the hind limb by using the forceps after the cervical vertebra. Peeled hind limbs were cut with surgical scissors and placed in α-MEM (Minimum Essential Medium Alpha) without serum. Tweezers were used to separate the bones in the muscle and transfer them to fresh α-MEM. 500 μl of α-MEM was put into a 1 ml syringe, and a syringe needle was inserted into the spinal cord of the center of the leg bone and sprayed twice to extract bone marrow cells. Bone marrow macrophages were isolated from the extracted bone marrow cells using Histopaque (Sigma, MO, USA). Isolated mouse bone marrow macrophages were cultured in α-MEM containing 1% Antibiotic-Antimycotic solution, 10% fetal bovine serum (FBS), and 30 ng / ml macrophage-colony stimulating factor (M-CSF). It was.

Compound isolated from peach PS -1 (2α, 3α- dihydroxyolean -12- en -28- oic acid )and PS -2 (2α, 3α-dihydroxyurs-12-en-28-oic acid Cytotoxicity measurement

In order to confirm the cytotoxicity against the mouse bone marrow macrophages of the peach active ingredient, the experiment was performed as follows (Wattel A. et al., J Cell) Biochem . , 92 (2): 285-295 (2004). Mouse bone marrow macrophages (1x10 ⁴ cells) were placed in each well of a 96-well plate and 1% Antibiotic-Antimycotic Solution, 10% Fetal Bovine Serum (FBS), and macrophage-colony (M-CSF) stimulating factor) 30 ng / ml, peach active ingredient PS-1 (2α, 3α-dihydroxyolean-12-en-28-oic acid), or PS-2 (2α, 3α-dihydroxyurs-12-en-28-oic acid) was incubated in α-MEM. Each compound was dissolved in DMSO (dimethyl sulfoxide) and diluted with 10% FBS-α-MEM to a concentration of 0, 2, 10 and 20 μM. Mouse bone marrow macrophages were replaced with fresh medium every other day for 5 days at 37 ° C, 5% CO ₂ After incubation in a cell incubator, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) solution was added to a concentration of 5 mg / ml for each well. After incubation at 37 ° C. for 4 hours, the medium and MTT solution were completely removed and 200 μl of DMSO was added per well. After 20 minutes the absorbance was measured at 570 nm. Cell viability was calculated as a percentage of the absorbance of the experimental group (well with the medium containing each peach active ingredient) relative to the absorbance of the control group (well treated only medium).

In the bone marrow macrophages, PS-1 (2α, 3α-dihydroxyolean-12-en-28-oic acid) and PS-2 (2α, 3α-dihydroxyurs-12-en-28-oic acid), active ingredients, , 2, 10 and 20 μM did not affect the cell viability of the mouse bone marrow macrophages when added (see Figure 7 and Table 1).

PS -1
(μM) Absorbance
(570 nm) Cell survival rate
(%) PS -2
(μM) Absorbance
(570 nm) Cell survival rate
(%) 0 0.831 100 0 0.999 100 2 0.828 99.63 2 0.981 98.20 10 0.810 97.47 10 0.985 98.60 20 0.822 98.96 20 0.970 97.10

Osteoclast Formation of Peach Active Ingredients Inhibition  Measure

In order to confirm the inhibitory effect on osteoclast formation from mouse bone marrow macrophages of the active ingredient of the peach, the experiment was carried out as follows according to the method described in the literature (Park EK. Et al., Biochem Biophys Res Commun . 325 (4): 1472-1480 (2004). The isolated mouse bone marrow macrophages were placed in a 96-well plate with 1 × 10 ⁴ per well, RANKL (Receptor Activator for Nuclear Factor κ B Ligand, 100 ng / ml), M-CSF (macrophage colony stimulating factor, 30 ng / ml ) And 10% FBS-α-MEM containing each peach active ingredient. Each peach active ingredient was incubated in 10% FBS-α-MEM containing 0, 2, 10 or 20 μM, respectively. Only 10% FBS-α-MEM was added to the control group. Every two days, the medium was exchanged with fresh medium, and cultured for 5 days, followed by staining using a TRAP assay kit (tartarate resistant acid phosphatase assay kit; Sigma, MO, USA) to confirm the formation of osteoclasts. The number of cells containing three or more nuclei, which are characteristic of osteoclasts, was measured using an optical microscope.

As a result, osteoclasts were not produced in mouse bone marrow macrophages not treated with RANKL, whereas the number of osteoclasts was significantly increased in mouse bone marrow macrophages treated with RANKL (p <0.0001) (see FIG. 8 and Table 2-3). ). When mouse bone marrow macrophages were treated with peach active ingredients, PS-1 (2α, 3α-dihydroxyolean-12-en-28-oic acid) and PS-2 (2α, 3α-dihydroxyurs-12-en-28 -oic acid) significantly inhibited the formation of RANKL-induced osteoclasts (see FIG. 8 and Table 2-3).

PS -1 (μM) Number of osteoclasts Osteoclast formation inhibition rate (%) P value 0 255.7 ± 24.3 - - 2 274.0 ± 13.1 0 - 10 42.0 ± 15.1 83.6 0.0002 20 0.3 ± 0.6 99.9 0.0001

PS -2 (μM) Number of osteoclasts Osteoclast formation inhibition rate (%) P value 0 255.7 ± 24.3 - - 2 275.0 ± 10.2 0 - 10 147.3 ± 21.1 42.6 0.004 20 0 100 0.0001

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (12)

A compound of formula 3 isolated from a peach extract; Pharmaceutically acceptable salts thereof; Or a pharmaceutical composition for the prevention or treatment of osteoporosis comprising a solvate or hydrate thereof as an active ingredient:
(3)

A compound of formula 3 isolated from a peach extract; Salts thereof; Or a food composition for preventing or improving osteoporosis comprising a solvate or hydrate thereof as an active ingredient:
(3)

delete delete delete delete The composition of claim 1 or 2, wherein the compound of Formula 3 inhibits the formation of osteoclasts.
8. The composition of claim 7, wherein the composition inhibits the formation of osteoclasts induced by a receptor activator of NFκB ligand (RANKL).
delete delete Functional food for the prevention or improvement of osteoporosis comprising the composition of claim 2.
The functional food according to claim 11, wherein the functional food is a powder, granule, tablet, capsule or beverage.

Images