KR101249930B1 - Compositions comprising extracts of Sorbus commixta and Geranium nepalense for inhibiting osteoclastogenesis and stimulating chondrogenesis - Google Patents

Abstract

The present invention relates to a composition for the prevention, improvement or treatment of bone and cartilage tissue damage diseases containing rowan extract or local herb extract or a mixture thereof as an active ingredient. Since the composition of the present invention exhibits the inhibitory activity of osteoclasts and / or promotes the differentiation of chondrocytes, and thus has a protective and regenerative effect on bone tissue or cartilage tissue, for the prevention or treatment of diseases associated with cartilage and bone damage. It can be widely applied for the purpose.

Description

Compositions comprising extracts of Sorbus commixta and Geranium nepalense for inhibiting osteoclastogenesis and stimulating chondrogenesis}

The present invention relates to a pharmaceutical composition for inhibiting and treating bone and cartilage tissue damage, comprising rowan extract (SC), local herb extract (GN) or a mixture thereof (SG-II) as an active ingredient. The pharmaceutical composition of the present invention inhibits the differentiation of osteoclasts and promotes the differentiation of chondrocytes and thus has a protective and regenerative effect on bone tissue or cartilage tissue, and thus is used for the prevention or treatment of diseases involving cartilage and bone destruction. It can be widely applied.

Bones support the soft tissues and weight of the body and surround internal organs to protect internal organs from external shocks. It is also an important part of the body that not only supports the muscles or organs but also stores calcium or other essential minerals, such as phosphorus or magnesium, in the body. Thus, bones of grown adults are balanced by dynamic and continuous regeneration of the process of producing and absorbing old bones and replacing them with new bones, which is called bone remodeling (Yamaguchi A. et al. , Tanpakushitsu Kakusan Koso., 50 (6Suppl): 664-669 (2005). This turnover of bone is essential for restoring and maintaining microscopic bone damage caused by growth and stress (Cohen-Solal M. et al., Therapie., 58: 391-393 (2003)). . In adults, about 10-30% of the skeleton is remodeled each year through bone resorption-bone remodeling.

In aggregate formation, two types of cells, osteoblasts that generate bone and osteoclasts that destroy bone, are involved and bone homeostasis is maintained in close association with each other. For example, osteoblasts regulate bone in the body by regulating the differentiation of osteoclasts responsible for bone resorption through the secretion of substances such as receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG). Maintain homeostasis. That is, when RANKL binds to RANK, an osteoclast progenitor cell surface receptor, osteoclast progenitor cells mature into osteoclasts, resulting in bone resorption, and when OPG binds to RANKL, binding between RANKL and RANK is blocked and inhibition of osteoclast formation ( Theill LE. Et al., Annu Rev Immunol., 20: 795-823 (2002); Wagner EF. Et al., Curr Opin Genet Dev., 11: 527-532 (2001)). Osteoclasts, which are formed from progenitor cells, absorb or destroy old bone, and release small amounts of calcium accumulated in the bone into the bloodstream to maintain body function (William J. et al., Nature., 423: 337342 (2003). )).

This metabolism is regulated by physical influences, hormonal systems and local factors, and various bone diseases occur when bone absorption exceeds bone formation and bone volume decreases below the limit by various factors. Osteoporosis is a condition in which the bone mass decreases below a certain level and the risk of fracture is continuously increased due to the degeneration of the microstructure of the bone tissue. The osteoporosis is a condition in which the minerals (particularly calcium) and the matrix that make up the bone are reduced. Occurrence of osteoclasts is greater than osteopathy, due to the loss of balance (Iqbal MM., South Med J., 93 (1): 2-18 (2000)). Osteoarthritis is the most common form of arthritis involving bones and cartilage, and excessively promotes osteoclast differentiation by inflammatory reactions. This increases the expression of bone resorption activating factors such as IL-1, TNF-α, MMP and eventually leads to bone loss and cartilage damage. Bone metastasis of cancer cells occurs frequently in breast cancer, prostate cancer or multiple myeloma (Kozlow W. et al., J Mammary Gland Biol Neoplasia., 10 (2): 169-180 (2005)), especially osteolytic bone metastases. Osteolytic metastasis is known to occur when breast cancer cells and the like activate osteoclasts (Boyde A. et al., Scan Electron Microsc., 4: 1537-1554 (1986)). Activated osteoclasts disrupt the balance of the microenvironment around bones, leading to bone-related diseases such as osteolysis and resulting pathological fractures, leukopenia, bone malformations, hypercalcemia, pain, and neurological compression syndrome (Roodman GD. , N Engl J Med., 350: 1655-1664 (2004).

In the past, studies on the treatment of diseases involving bone tissue damage have been conducted mainly on the metabolic abnormalities of bone minerals, that is, calcium and phosphorus, and no progress has been made in identifying the mechanism. In general, a diet containing calcium is recommended for the treatment and prevention of osteoporosis, and estrogen or vitamin D administration is recommended for postmenopausal women. In addition, bisphosphonate-based agents such as Fosamax and Actonel are attracting attention as new alternative therapeutics as bone resorption inhibitors that inhibit osteoclasts and induce death.

However, calcium adjuvant, widely used as a therapeutic agent for bone diseases, suppresses secretion of parathyroid hormone and prevents bone loss due to bone resorption, but it is known that individual differences in bone maintenance are severe (Heandy. RP principles of bone biology, Academic press , 1007-1017, 1996). In addition, hormone therapy with estrogen or calcitonin has been reported to increase bone density and reduce the incidence of rectal cancer, but side effects such as breast cancer, myocardial infarction and venous thrombosis have been reported (Nelson, HD et al., JAMA, 288: 872-881, 2002; Lemay, A., J. Obstet. Bynaecol. Can., 24: 711-7152-3). In addition, in the case of bisphosphonate preparations, the incidence of jaw bone necrosis, severe atrial fibrillation, incapacitation of bones or joints, or musculoskeletal pain is increasing year by year (Coleman RE., Br J Cancer, 98: 1736-). 1740 (2008) Therefore, there is a need in the art for the development of a new treatment for bone disease, which can effectively suppress bone resorption with less side effects.

Chondrocytes differentiate from mesenchymal cells during development to form cartilage tissue. Unlike other types of tissues, cartilage is composed of only one kind of chondrocytes, and the formed cartilage tissue remains as permanent cartilage tissue, such as articular cartilage, or functions as a template for tibia formation during development. Therefore, substances that promote chondrocyte differentiation may primarily contribute to bone mineral density by increasing the rate of bone (tibia) tissue formation.

Articular cartilage is the most heavily loaded tissue of human tissue and is easily exposed to damage caused by wear. Intraarticular chondrocytes play an important role in the maintenance of articular cartilage because they secrete extracellular matrix, and the decrease in the number or metabolism of chondrocytes due to aging is considered to be a major cause of osteoarthritis. Intra-articular chondrocytes are adherent cells that do not have blood vessels, nerves, and lymphoid tissues. Therefore, current medical methods have a very limited method of regenerating articular cartilage normally. In general, a method of regenerating damaged articular cartilage of a patient is often used by taking a portion of normal cartilage tissue and amplifying the chondrocytes in the appropriate culture medium to amplify the chondrocytes and transplanting them into the cartilage defect. To this end, there is a need for the development of substances for promoting the differentiation and proliferation of chondrocyted stem cells into chondrocytes.

Rowan ( Sorbus commixta , SN) is a deciduous broad-leaved small arborescent of the Rosaceae family, and is naturally distributed in deep mountain forests of 500-1200 m above sea level in Korea, China and Japan. The bark of rowan was called Mafi, which has been used for herbal protection, bronchitis, palsy gastritis and bone pain in kidneys in Korea. The physiological activities of rowan are known to inhibit diabetes and obesity, to inhibit antioxidant and photoaging effects, to inhibit vascular inflammation and to anti-arteriosclerosis, and the components include neosakuranin, lupeol and luphenone. (lupenone) is known, but studies on physiological activity in bone disease is insufficient.

Geranium nepalense (GN) is a perennial herb that belongs to the rat deciduous family. It is distributed throughout Korea, Japan, Taiwan, and China. Dry stems and leaves used for boils, raises, abdominal pain, constipation, etc. In particular, because it is known as a heterogeneous medicinal agent, it is also known as a dysentery. Antimutagenic and antioxidative effects have been reported as other activities, but the physiological activity in bone disease has not been disclosed or taught.

It is an object of the present invention to provide a method for the prevention or treatment of a safe and effective bone or cartilage tissue damage disease and other related diseases and a composition therefor. To this end, it was intended to provide a composition having an osteoclast inhibitory activity or a chondrocyte differentiation promoting activity and having an excellent prophylactic or therapeutic effect on the bone disease.

For this purpose, the present invention provides a composition having osteoclast differentiation inhibitory activity or chondrocyte differentiation promoting activity, comprising rowan (SC) or local herb extract (GN) or a mixture thereof (SG-II).

The present invention also provides a pharmaceutical composition for the prevention or treatment of diseases caused by excessive differentiation and activation of osteoclasts, and other diseases accompanied with bone or cartilage damage, including the rowan or R. herb extract or mixtures thereof .

The present invention also provides a method of preventing, treating or ameliorating the disease by administering an effective amount of the composition to a mammal.

The present invention also provides a method for producing a medicament for preventing or treating the disease, comprising an effective amount of the composition.

The present invention also provides a method for inhibiting osteoclast differentiation or promoting chondrocyte differentiation.

Hereinafter, the present invention will be described in detail.

The pharmaceutical composition of the present invention contains rowan extract, local herb extract or a mixture thereof as an active ingredient. The extracts significantly inhibit osteoclast differentiation (osteeogenesis) and at the same time significantly increase chondrogenesis, and thus can be used for the prevention and treatment of various bone diseases.

Specifically, rowan extract, local herb extract or a mixture thereof decreases concentration-dependently the activity of TAP (Tartrate-resistant acid phosphatase), a marker enzyme of osteoclasts, compared to cells not administered with the extract (FIGS. 2 and 3). ). Therefore, the composition of the present invention exhibits the effect of reducing the formation of osteoclasts and inhibiting bone resorption and bone destruction by osteoclasts. As used herein, the term "bone resorption" refers to a metabolic process in which osteoclasts break down bone by releasing strong acids and proteases into bone.

Accordingly, pharmaceutical compositions comprising rowan extract, local herb extract or mixtures thereof may be used for the prevention and treatment of bone diseases. The term "bone disease" is used herein in a broad sense, including diseases caused by overdifferentiation and activation of osteoclasts and all diseases that are accompanied by or are likely to be accompanied by damage to bone tissue or cartilage tissue. The bone disease is fracture, osteoporosis, osteoarthritis, degenerative cartilage and bone destruction disease, rheumatic disease, periodontal disease, bone tumor, bone damage caused by bone metastasis of cancer cells, Paget's disease, metabolic osteoarthritis, fibrous osteoarthritis, aplastic bone disease, Osteomalacia, bone malformations, rickets, leukemia, hypercalcemia, neuropathy syndrome, multiple myeloma, and the like.

The osteoporosis includes primary (primary) osteoporosis and secondary (secondary) osteoporosis. Primary osteoporosis refers to osteoporosis that does not ingest calcium and vitamin D properly or occurs mainly in postmenopausal women or older men. Secondary osteoporosis is caused by certain diseases or drugs. The latter causative diseases include hyperthyroidism, parathyroidism, Cushing's syndrome (adrenal cortical hormone secretion), early menopause, menopause caused by artificial surgery (ovarian resection), hypogonadism, chronic liver disease (cirrhosis of the liver), rheumatoid arthritis , Chronic renal failure, gastrectomy, and the like, drugs include steroids (corticosteroids), anticonvulsants (epilepsy drugs), heparin and the like.

In bone disease caused by bone metastasis of the cancer cells or cancer tissue, the cancer cells or cancer tissue, for example, gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, rectal cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, Colon cancer, cervical cancer, endometrial cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, leukemia, non-Hodgkin lymphoma, parathyroid cancer and ureter cancer cells or tissues.

In addition, rowan extract, local herb extract and mixed extract of rowan and local herb promotes the differentiation of cartilage progenitor cells into chondrocytes in a concentration-dependent manner to show the effect of cartilage tissue regeneration (FIG. 4).

Accordingly, the pharmaceutical composition comprising the extract can be used as a medicament for regeneration or protection of cartilage tissue by promoting the production of chondrocytes. Specifically, the composition of the present invention can be used for the prevention or treatment of cartilage damage, joint damage due to trauma and other joint diseases or conditions involving cartilage destruction, osteoarthritis (degenerative arthritis), rheumatoid arthritis.

As used herein, the terms “mountain ash extract” and “local herb extract” are meant to be obtained by extracting from various organs or parts (eg, leaves, flowers, fruits, bark and stems, etc.) of rowan or local grass.

The term “extract” is meant to include not only the extraction result obtained by treating the rowan or extract with an extraction solvent, but also a workpiece formulated (eg, powdered) for administration to the animal of the mountain ash or vinegar itself. In addition, the "extract" has a meaning commonly used as a crude extract (crude extract) in the art as described above, but broadly also includes an additional fraction (fractionation) of the extract. That is, the extract of rowan or local herb, as well as obtained by using the above-mentioned extraction solvent, it includes also obtained by further applying the purification process. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, a fraction separated by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), and the like, Fractions obtained through various purification methods.

When the extract of mountain ash or local herb used in the composition of the present invention is obtained by treating the extract with mountain ash or local herb, various extracting solvents including a polar solvent and a nonpolar solvent may be used. Suitable polar solvents include water, alcohols (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol), acetic acid , Dimethyl-formamide (DMFO) and dimethyl sulfoxide (DMSO). Suitable nonpolar solvents include acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1-pentene , 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane , 1,2-dichloroethane, anneal, diethylamine, ether, carbon tetrachloride and THF.

More preferably, the extraction solvent used in the present invention is water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, acetone, ethyl acetate , Chloroform, butyl acetate, 1,3-butylene glycol, hexane and diethyl ether. Most preferably, the extract of the present invention is obtained by treating water, butanol, ethyl acetate, or a combination thereof, in rowan or local grass.

Extraction may be carried out at 80 to 120 ° C., preferably at about 100 ° C., for 2 to 6 hours, preferably for about 2 hours, but the extraction temperature and extraction time may be used for the herbal medicines (rowan or local grass) and It may be changed depending on the amount of the extraction solvent.

In addition to the above process, it may further comprise the step of concentrating and / or drying as necessary, the concentration and / or drying may be carried out by conventional methods, for example, vacuum distillation and freeze drying or spray drying. have. Preferably, the mountain ash and / or vinegar extract used in the present invention may be in the form of a powder dried by vacuum concentration and freeze drying.

The pharmaceutical composition obtained by the method comprises 0.1 to 50% by weight of rowan extract, local herb extract or a mixture thereof based on the total weight of the composition.

In addition, in the mixture of rowan extract and local herb extract, the blending ratio of each extract is preferably in the range of 1:10 to 10: 1, preferably 1: 2 to 2: 1 by weight. More preferably, the mixture contains rowan extract and local herb extract in a weight ratio of 1: 1.

In the compositions according to the invention, the pharmaceutical dosage forms of the extracts can also be used in the form of their pharmaceutically acceptable salts, and can also be used alone or in the form of combinations or aggregates with other pharmaceutically active compounds. have.

A pharmaceutical composition comprising the rowan and / or Cannabis extract according to the present invention may be formulated in conventional pharmaceutical formulations known in the art. The formulations may be formulated and administered in any of the formulations, including but not limited to oral, injectable, suppository, transdermal, and non-administrative formulations, preferably liquids, suspensions, syrups, powders, granules Oral dosage forms such as tablets, capsules, pills, or excipients.

In one embodiment, the composition of the present invention is a solid preparation for oral administration, and may be in the form of tablets, pills, powders, granules, capsules and the like. Such solid preparations may include at least one or more pharmaceutically acceptable excipients such as diluents, glidants, binders, disintegrants, sweeteners, stabilizers, and preservatives in the rowan and / or local herb extracts. Specifically, lactose, corn starch, gelatin, calcium carbonate, soybean oil, microcrystalline cellulose, or mannitol, magnesium stearate or talc as a lubricant, and polyvinylpyrrolidone or hydroxypropyl cellulose as a binder may be used. have. In addition, as a disintegrant, carboxymethyl cellulose calcium, sodium starch glycolate, potassium polyacrylic acid, or crospovidone, sweetener as white sugar, fructose, sorbitol, or aspartame, stabilizer as carboxymethyl cellulose sodium, beta-cyclodextrin, As lead, or xanthan gum, and preservatives, methyl paraoxybenzoate, propyl paraoxybenzoate, or potassium sorbate may be used.

In another embodiment, the composition of the present invention is a liquid preparation for oral, and may be in the form of a suspension, a liquid solution, an emulsion, a syrup. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients may be included, for example wetting agents, sweetening agents, fragrances, preservatives and the like. In addition to the above ingredients, in order to enhance the taste as well-known additives, natural flavors such as plum, lemon, pineapple, herbal flavor, natural pigments such as fruit juice, chlorophyllin, flavonoids, fructose, honey, sugar alcohol, sugar Sweetening ingredients such as, or may be used by mixing an acidulant such as citric acid, sodium citrate.

In another embodiment, the pharmaceutical compositions of the present invention may be a preparation for parenteral administration and include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. Injectables may be prepared using a physiologically suitable buffer such as, but not limited to, Hank's solution, Ringer's solution, and saline buffer. In addition, non-aqueous solvents and suspending agents may be used propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like, and may further include stabilizers, dispersants, and the like. The compositions of the present invention may also be prepared in powder form and used with a suitable carrier such as sterile water before use. When formulated as a suppository, the base may be, but is not limited to, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The rowan and / or field herb extracts of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by a variety of routes. All modes of administration can be expected, for example oral, intraarticular, intravenous, intramuscular, subcutaneous, transdermal, rectal, intranasal, intraperitoneal, and the like. The composition may also use other methods known in the art, such as those described in the latest edition of Remington's Pharmaceutical Science.

Suitable dosages and timing of administration of the compositions according to the invention will vary depending on the method of formulation, the subject's age, sex, weight, degree of disease symptom, rate of excretion and sensitivity to reaction, route of administration and duration of administration, but will not affect the treatment desired by those skilled in the art. Effective dosages may be appropriately selected. For example, it may be administered in several divided doses so as to be 0.1 mg / kg to 10 g / kg, preferably 100 mg / kg to 1 g / kg, per adult, per day.

The composition of the present invention may be used alone as a therapy, but may be used in combination with surgery, hormonal therapy, drug therapy, methods using biological response modifiers, diet and the like.

In another aspect of the present invention, there is provided a method of improving bone density by administering to a subject a composition comprising a rowan and / or vinegar extract. The subject may be a variety of mammals, including humans. Inhibitory activity of osteoclasts to inhibit osteoclast differentiation of rowan or local herb extracts can reduce bone resorption, and improvements in bone density include individuals with potential bone loss, including regeneration of lost bones (eg, postmenopausal women, the elderly, Prevention of bone mineral density in patients undergoing hormonal therapy).

In still another aspect of the present invention, there is provided a method for the preparation of a medicament for preventing or treating a bone or cartilage tissue damage disease, comprising an extract of rowan and / or zinnia. The method comprises the steps of extracting rowan or Cultivation using a suitable extraction solvent, preferably water; Filtering and concentrating under reduced pressure; And optionally, mixing an effective amount of the extract obtained therefrom with one or more of pharmaceutically acceptable carriers, excipients, and diluents. In the case of the mixture of rowan and local herb extract, the method further comprises the step of mixing the extract concentrated in the above method, preferably in a weight ratio of 1: 1.

In another aspect of the invention, the invention provides a method of promoting differentiation of chondrocytes comprising contacting an effective amount of mammalian chondrocytes and rowan extract, local herb extract or a mixture of rowan and local herb extracts. do.

In one embodiment, the contacting step may be performed by a method of adding the composition to the culture medium of chondrocytes collected and separated from the damaged articular cartilage tissue.

The differentiated chondrocytes produced in vitro by the method of the present invention can be usefully used to treat cartilage damage diseases by biological methods through transplantation of cells or tissues. More specifically, chondrocytes produced by the method of the present invention are directly injected into the joint of a patient according to a known method as an active ingredient of a cell composition for cell replacement therapy, or with a scaffold after three-dimensional culture. (Kim, G. et al, J. Vet. Med. Sci., 66: 263, 2004, Lee, JW et al., Yonsei Med. J., 30:41, 2004).

In another embodiment, the contacting step may be performed by a method of directly injecting a composition comprising rowan extract or mixed extract into mammalian cartilage tissue. For example, in the 'regenerative cell therapy' which induces the regeneration of chondrocyte tissue in the body by injecting healing stem cells or healing regenerative cells extracted and cultured in abdominal fat and the like into the affected area, the composition of the present invention is Injecting together may promote differentiation of the cells into chondrocytes. Cartilage damage diseases that can be treated using chondrocytes produced by the method of the present invention include, but are not limited to, degenerative arthritis, rheumatoid arthritis, and joint damage due to trauma. The differentiated chondrocytes are less likely to differentiate into other cells, such as osteocytes, and because they use cells derived from autologous cells, there is no immunological rejection reaction, and thus there is an advantage that it can be used more safely for treatment.

In a further aspect of the invention, the present invention provides a health functional food composition for the prevention or amelioration of bone disease, comprising rowan extract, local herb extract or a mixture thereof and a food acceptable additive.

As used herein, "health functional food" refers to a food prepared and processed using raw materials or ingredients having functional properties useful for the human body, and "functionality" refers to nutrients or physiological effects on the structure and function of the human body. It means to consume for the purpose of obtaining a useful effect for health use such as.

The composition comprising the extract of mountain ash or vinegar of the present invention may be formulated as a health functional or beverage, or added to various foods for the prevention and improvement of diseases accompanied with bone or cartilage tissue damage.

Such health functional food compositions may be formulated in the form of conventional health functional foods known in the art in the same manner as the pharmaceutical compositions. The health functional composition may be prepared, for example, as a powder, granules, tablets, capsules, suspensions, emulsions, syrups, solutions, extracts, teas, jellies, beverages, and the like. As the food acceptable acceptable additive, any carrier or additive known to be usable in the art may be used to prepare a formulation to be prepared.

When the composition of the present invention is used in addition to other foods, foodstuffs that can be added include, for example, beverages, meat, candy, chocolate, confectionery, gum, tea, vitamin complexes, alcoholic beverages, and health supplements.

In this case, the amount of the composition in the food or beverage, in general, in the case of the health functional food composition of the present invention can be added to 0.01 to 15% by weight, preferably 1 to 5% by weight of the total functional food composition, In the case of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

In the case of the health functional beverage composition of the present invention, there is no particular limitation on the liquid component except for containing the extract as an essential ingredient in the indicated ratios, and various flavors or natural carbohydrates may be added as additional ingredients, as in general beverages. Can be. Examples of natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as conventional sugars such as dextrin, cyclodextrin and the like and sugar alcohols such as xylitol, sorbitol, erythritol and the like. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to this, usefully, natural flavors (tauumatin, stevia extracts such as rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) may be used as flavoring agents. In addition, the composition of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective properties Colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages.

These components can be used independently or in combination. The proportion of such additives is not limited but is generally selected in the range of 0 to 20% by weight relative to the total weight of the composition of the present invention.

All technical terms used in the present invention are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention, unless otherwise defined. Also described herein are preferred methods or samples, but similar or equivalent ones are included in the scope of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

The composition according to the present invention comprises a mountain ash or local herb extract, or a mixture thereof in a predetermined amount or more, has a remarkable effect on the osteoclast inhibition or chondrocyte differentiation promoting activity to the extent that those skilled in the art can not predict, made of natural substances There are few side effects to it, which is desirable. Therefore, the composition according to the present invention can be used for the manufacture of a medicament or dietary supplement that can be used for the prevention, improvement and treatment of diseases caused by excessive differentiation or activation of osteoclasts or bone or cartilage tissue damage diseases. have.

In addition, the composition of the present invention ( in vitro) vitro) or in vivo (in In vivo ) can be used to promote the differentiation of chondrocytes, can be used for the purpose of regeneration of cartilage defects and the manufacture of artificial joints.

1 is a graph showing the effect of rowan extract, local herb extract and 1: 1 mixture thereof according to the present invention on ALP activity in MG-63 cell line (SC; rowan extract treatment group, GN; local herb extract) Treatment group, SG-II; 1: 1 mixture of rowan extract and local herb extract, EST; beta-estradiol treatment group). (*: p <0.05)
Figure 2 is a graph comparing the effect on the activity of TRAP when the rowan extract, local herb extract and 1: 1 mixture thereof according to the present invention in Raw264.7 cell line by concentration (NC; negative control, PC Positive control (RANKL 50 ng / ml treatment), SC; rowan extract treatment group, GN; local herb extract treatment group, SG-II; 1: 1 mixture of rowan extract and local herb extract).
Figure 3 is a micrograph showing the degree of differentiation of Raw264.7 cells into osteoclasts when incubated with a 1: 1 mixture of rowan extract and rowan and local herb extracts (10, 250 μg / ml).
Figure 4 is a graph comparing the degree of differentiation into chondrocytes when the rowan extract, local herb extract and 1: 1 mixture thereof according to the present invention to the ATDC5 chondrocyte precursor cells induced differentiation by concentration. (*: p <0.05)
FIG. 5 is a photograph showing rowan extract, local herb extract, and 1: 1 mixture thereof in ATDC5 cartilage precursor cells by concentration, and staining with Alciain blue to differentiate into chondrocytes.

Hereinafter, the present invention will be described in more detail by the following Preparation Examples and Examples. However, these examples are only for the understanding of the present invention, and the scope of the present invention in any sense is not limited thereto.

Materials and preparation

Rowan ( Sorbus commixta , SC) were purchased from OmniHub (Youngcheon, South Korea), and local grass ( Geranium nepalense , GN) from Daeram Pharmaceutical (Seoul, South Korea).

DMEM, F-12 and -MEM culture medium, fetal bovine serum (fetal bovine serum, FBS), penicillin-streptomycin (PS) were purchased from Gibco (NY, USA), LabAssay ^TM ALP kit Wako (Osaka, Japan) The receptor activator of NF-κB ligand (RANKL) was purchased from PeproTech (NJ, USA) respectively. Other substances such as β-estradiol, transferrin, sodium selenite, bovine insulin, sodium nitrite and alkyan blue 8GS are described in Sigma-Aldrich Chemical Co. It was purchased from (MO, USA) and used.

rowan  And Local grass  Preparation of extract

To obtain the extract, after washing each sample (fruit for rowan, leaf for local grass), distilled water of 10 times the weight of each sample was added and reflux extraction for 2 hours at 100 ℃, the filtration process was repeated three times. Then, the extract obtained by filtration was concentrated under reduced pressure and lyophilized. Yield of each sample was 8.10% of rowan extract, 13.15% of local herb extract, and mixed samples (MIX) were prepared by mixing each of the rowan and local herb extract samples in a ratio of 1: 1.

Statistical processing

The above experiments were repeated three times each, and the experimental results were obtained. All data were calculated as a percentage of the control group and expressed as mean ± standard deviation. The experimental groups were compared and analyzed using Student's t test, and the significance level of the statistical method was set to p-value <0.05.

Example  1.basic phosphatase ( Alkaline phosphatase , ALP ) Activity and cell viability measurements

ALP is a glycoprotein produced by osteoblasts and is a cytochemical marker that is used as an indicator of bone formation. It is known to be activated by bone morphogenetic protein (BMP), a regulator that induces bone and cartilage formation and regeneration, to promote osteoblast differentiation and stimulate collagen synthesis (Wozney, JM, et al. , "Novel regulators of bone formation: molecular clones and activities", Science, pp. 1528-1534, 1988).

To investigate the effects of rowan, locust grass and 1: 1 mixed samples on ALP activity of osteoblasts, each sample was treated with MG-63 cell line for 48 hours, and then ALP was a colorless p-nitrophenylphosphate. ALP activity in cells was measured using the principle of decomposing to phosphoric acid and yellow p-nitrophenol.

Way

One) ALP  Active measurement

Human osteoblast-like MG-63 cell line (Seoul, Korea Cell Line Bank) was cultured in a 5% CO ₂ incubator at 37 ° C. using DMEM medium containing 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin. Incubated. When the cells were grown to about 85 ± 5% in the culture vessel, trypsin-treated cells were separated, and the 6-well culture vessel was dispensed to contain 1.5 × 10 ⁵ cells per each well. After 24 hours, each sample (10, 100 and 250 μg / ml) was treated together and incubated for 48 hours. At this time, β-estradiol (10 μM), which is known to increase ALP activity, was used as a positive control.

The cells were washed with PBS and treated with trypsin to remove, and 50 μl of 0.2% Triton X-100 was added thereto, and the cells were lysed using an ultrasonic extractor at 4 ° C. for 3 minutes. Cell lysates were centrifuged at 14000 rpm for 20 minutes at 4 ° C., supernatants were taken to quantify proteins and ALP activity was measured. ALP activity measurement experiments were performed using the LabAssay ^™ ALP kit, and the change in absorbance at 405 nm was measured. Intracellular proteins were also quantified and quantified by ALP / protein to determine the relative activity of the samples.

result

Each sample did not affect ALP activity regardless of concentration (FIG. 1). However, β-estradiol, a positive control, increased ALP activity by 138.3 ± 5.8% after 10 μM treatment.

Example  2. Tartrate Resistant acid phosphatase ( Tartrate Resistant Acid  Phosphatas, TRAP A) positive staining

TRAP is an enzyme secreted by osteoclasts and is a cytochemical marker that is used to measure the degree of differentiation of osteoclasts. During bone resorption, secretion is increased and involved in the breakdown of bone matrix (Shin Chung-ho, "Clinical Utility of Biological Markers of Bone Turnover in Children and Adolescents", Journal of Korean Endocrinology, Vol. 6, No. 1, pp. 4-16, 2001.), ATP, nitrophenyl phosphate in the presence of high activity. On the other hand, RANKL is known to be an essential cytokine for osteoclast differentiation with M-CSF because it has an effect of increasing osteoclast activity of osteoclasts and enhancing cell viability.

In this experiment, we examined the effects of rowan, vinegar extract, and 1: 1 mixed samples on osteoclast differentiation of RAW 264.7 cell line derived from mouse macrophage, and treated each sample with RANKL for 6 days. Treated together and observed by TRAP staining.

Way

The raw monocyte / macrophage Raw 264.7 cell line (Seoul, Korea Cell Line Bank) was treated with 5% CO ₂ at 37 ° C. using α-MEM medium containing 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin. Cultured in an incubator. When the cells grew to about 85 ± 5% in the culture vessel, trypsin-treated cells were separated, and the 96-well culture vessel was dispensed to include 1 × 10 ³ cells in each well. After 24 hours, 50 ng / ml RANKL was treated to promote differentiation of Raw 264.7 cells into osteoclasts, and each sample was incubated together for 6 days.

After 6 days of sample treatment, the medium of cells was removed for TRAP staining, and then the cells were fixed for 10 minutes with a fixing solution (citric acid solution, acetone, 37% formaldehyde). Remove 1 minute after adding 0.1% Triton X-100, remove substrate solution (50 μl Fast Garnet GBC Base solution, 50 μl sodium nitrite solution, 4.5 ml distilled water, 50 μl naphthol AS-BI phosphate solution, 200 μl acetate solution, 100 μl) The cells were stained using tartrate solution) and reacted at 37 ° C. for 40 minutes to stain the cells. The osteoclasts differentiated under a microscope were observed and TRAP activity was measured by counting the number of stained multinucleated cells (cells with three or more nuclei).

result

As osteoclasts differentiate, cell morphology changes and the number of nuclei increases. Thus, as the number of osteoclasts that change shape and increase nucleus decreases, the drug shows a greater effect. In this experiment, the number of cells with three or more nuclei was counted to predict the degree of osteoclast differentiation and the effects of drugs in each sample.

As a result, all three samples showed a tendency to decrease TRAP activity in a concentration dependent manner. The rowan inhibited TRAP activity by 43.0 ± 19.9% at 250 μg / ml compared with the control group.In particular, the 1: 1 mixed sample of rowan and local herb at 23.0 ± 2.9% and 95.0 at concentrations of 100 and 250 μg / ml. It was confirmed that the effect was very excellent from the reduction of ± 3.8%. Local herb extract showed a high reduction of TRAP activity at low concentrations of 10 μg / ml and almost completely reduced TRAP activity at concentrations of 100 μg / ml (FIG. 2).

Example  3. Alcian blue ( Alcian - blue ) Staining

The following studies were conducted to investigate the effects of extracts of rowan, locusts, and 1: 1 mixed samples on the promotion of chondrocyte differentiation. In the case of alcian blue staining, when the differentiation into chondrocytes was performed, the principle that the collagen was stained and became blue was used.

Way

ATDC5 cells (purchased from Riken Cell Bank, Japan), which are precursors of chondrocytes, can be obtained from 5% FBS, 100 U / ml penicillin and 100 μg / ml streptophore in a medium containing 1: 1 mixed DMEM and F-12. Incubated in a 5% CO ₂ incubator at 37 ℃ using a medium mixed with mycin. When cells were filled in 75 ± 5% of the culture vessels, the cells were detached by trypsin treatment, and the cells were divided into 24-well culture vessels containing 2 × 10 ⁴ cells per well. After 24 hours, cell differentiation was induced for 14 days in medium containing 10 μg / ml transferrin and 3 × 10 ⁻⁸ M sodium selenite, during which time each sample was at a concentration of 10, 100 and 250 μg / ml. Treated together. 10 μg / ml bovine insulin was used as a positive control.

After 14 days, the medium of cells was removed and washed with 1 × phosphate buffered saline (PBS) to perform alkian-blue staining, then the cells were fixed with 95% methanol and 16 hours with 1% alcian-blue 8GS. Stained. After removing the dyeing solution, washed three times with 3% acetic acid for 30 seconds, and observed and photographed with a microscope to check the degree of staining. After staining with 10% acetic acid, the absorbance was measured at 650 nm.

result

As a result, rowan extract and mixed samples promoted chondrocyte differentiation in a concentration-dependent manner (Fig. 4). The rowan increased chondrocyte differentiation by 83.7 ± 2.5% compared to the control at a concentration of 250 μg / ml, which was higher than the positive control treated with bovine insulin (31.2 ± 2.0%). Local herb extract induced chondrocyte differentiation by 14.1 ± 1.4% (compared to negative control) at 100ug / ml. In addition, the 1: 1 mixed sample of rowan and local herb induced 31.9 ± 1.6% of chondrocyte differentiation at a concentration of 100 ug / ml compared to the control group, and showed higher values when the rowan extract alone or local herb extract alone was used. Chondrocyte differentiation was induced 70.92 ± 1.23% compared to the control at a concentration of 250 μg / ml.

Hereinafter, some formulation examples of medicinal and health functional foods including the composition of the present invention are presented, but are not limited thereto. The pharmaceutical compositions of the invention may be prepared in the form of granules, suspensions, solutions, capsules, tablets and the like, in any manner known in the art to obtain the best possible formulation.

Formulation example  1. Oral Formulations-Preparation of Tablets

Constituent Content (mg / tablet) Extract of the Invention 200 Lactose 150 Hydroxypropylcellulose 15 Carboxymethylcellulose Calcium 20 Magnesium stearate 5 glycerin 2 Hydroxypropylmethylcellulose 6 Titanium oxide 2

The extract, cellulose derivative and lactose of the present invention are mixed. Magnesium stearate and glycerin are added to the obtained premix and mixed. The final mixture is compressed into tablets according to a conventional tablet preparation method and finally coated with titanium oxide. Take 1 tablet three times a day.

Formulation example  2. Oral preparations-preparation of granules

Constituent Content (mg / Sachet ) Extract of the Invention 400 Excipient Proper amount Microcrystalline cellulose 65 Corn starch 15.5 Sorbitol 2250 Polyvinylpyrrolidone 47.5 Citric acid 10 Aspartame 32 Flavor 180

The extract, microcrystalline cellulose and corn starch of the present invention are mixed. The mixture is kneaded using a binder solution consisting of water and polyvinylpyrrolidone. Once the mixture is wet, it is granulated through a 2 mm sieve. The granules are dried and forced to pass through a 0.8 mm sieve and divided into four. The granulated powder obtained is mixed with sorbitol, citric acid, aspartame and a suitable flavoring agent. The granules are filled in a sachet.

Formulation example  3. Formulations for injection injectable formulation Manufacturing

Constituent content( mg ) Extract of the Invention 10 Sodium metabisulfite 3.0 Methyl paraben 0.8 Propylparaben 0.1 Sterile Distilled Water for Injection Appropriate amount to 2 ml

The ingredients are mixed to prepare a final injection volume of 2 ml by conventional means and filled in ampoules. The final solution should be sterile and nonpyrogenic. Alternatively, the injectable preparation may be prepared in lyophilized powder and dissolved in the injectable medium prior to use.

Formulation example  4. Health drink  Produce

Constituent Content (g) Extract of the Invention One Citric acid One oligosaccharide 100 Plum concentrate 2 Taurine One Purified water Proper amount up to 900 ml

After mixing the components according to the conventional healthy beverage production method, and then stirred and heated at 85 ℃ for about 1 hour, the prepared solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated. In this formulation example, although the component suitable for a favorite drink was mixed and mixed as a preferable ratio, the compounding ratio may be modified arbitrarily.

Claims (10)

Fracture, Osteoporosis, Periodontal Disease, Bone Tumor, Paget's Disease, Osteomalacia, Multiple Myeloma, Leukemia, Rickets, Fibrous Osteitis, Amorphous Bone Disease, Metabolic Bone Disease, Cancer Cell Bone A pharmaceutical composition for preventing or treating a disease or condition selected from the group consisting of injury, osteolysis, bone malformation, hypercalcemia, and neurocompression syndrome. The pharmaceutical composition of claim 1, further comprising a local herb extract. delete delete According to claim 1 or 2, wherein the extract is water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, a mixed solvent of the lower alcohol and water, acetone, ethyl acetate, chloroform, butyl acetate, butylene glycol, A pharmaceutical composition which is extracted using an extraction solvent selected from the group consisting of propylene glycol, dichloromethane, hexane, diethyl ether and mixtures thereof. delete Comprising the mountain ash extract as an active ingredient, a health functional food composition for bone density improvement. The health functional food composition of claim 7, further comprising a local herb extract. A health functional food for preventing or ameliorating bone disease, comprising the composition of claim 7 or 8 and a food supplement acceptable food supplement. The health functional food of claim 9 in the form of a powder, granule, tablet, capsule, pill, suspension, emulsion, syrup, liquid, extract, tea, jelly, or beverage.

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