KR101659659B1 - A pharmaceutical composition for preventing or treating bone diseases comprising Sinomenium acutum extract - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a health functional food composition for preventing or treating osteoporosis comprising Sinomenium acutum extract, a fraction thereof, a compound isolated therefrom, or a pharmaceutically acceptable salt thereof, as an active ingredient More specifically, the composition of the present invention has an effect of inhibiting the differentiation and activity of osteoclasts, and can inhibit osteopathy due to bone loss, and thus can be usefully used as a pharmaceutical composition for preventing or treating these diseases .

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for prevention or treatment of osteoporosis,

The invention abandonment that can be useful in the prevention or treatment of bone disease (Sinomenium acutum extract and the use of the compounds isolated therefrom.

The bone tissue constitutes the cartilage and skeletal system, and functions as a support and a muscle attachment by the mechanical function, and functions to protect the living organ and the bone marrow. It also functions to preserve calcium and phosphorus ions to maintain homeostasis. It is a very dynamic tissue that maintains homeostasis by breaking down little by little every day and forming new bone in proportion to the amount disintegrated. Specifically, the bone is repeatedly circulated for about 120 to 150 days including osteoclast-induced bone resorption, osteoblast-induced bone formation, and resting period. In healthy adults, bone resorption and bone formation are strictly controlled, and the total bone mass remains almost constant, with little change.

Osteoblasts originate from mesenchymal stem cells. Mineralization such as calcium formation by osteoblast differentiation not only maintains bone strength but also plays an important role in the homeostasis of calcium and hormone metabolism throughout the body. Calcium formation by osteoblast differentiation is regulated by vitamin D and parathyroid hormone. Bone morphogenesis by osteoblast differentiation induces bone morphogenetic protein (BMP), Wnt, MAP Related to the differentiation of osteoblasts by the cross-talk of various signal transduction systems such as kinase, calcineurin-calmodulin kinase, NF-κB and AP-1. phosphatase, and ALP are synthesized in the early differentiation stage and then osteopontin, osteocalcin, type 1 collagen, etc. associated with mineralization are synthesized (Pittenger, MF et al., Multilineage Potential of Adult Human Mesenchymal Stem Cells, Science, 1999, 284: 143-147). That is, the compounds that promote the activity of the alkaline phosphatase promote bone cell differentiation and can be targets of therapeutic agents for bone diseases.

On the other hand, osteoclast is a polynuclear giant cell that melts calcified cartilage and bone tissue to allow bone to grow. The osteoclasts are differentiated by RANKL (receptor activator of NF-κB ligand) secreted from osteoblasts and M-CSF (macrophage colony stimulating receptor) binding to receptors on the osteoclast surface.

When the balance between the osteoblast and osteoclast is broken, systemic or local bone disease is caused. Most of these diseases are known to be caused by an increase in the number and activity of osteoclasts. The diseases caused by the increased activity of osteoclasts include fractures, rheumatoid arthritis, primary bone tumors, bone metastasis of cancer, and the like. In addition, the activity of osteoclasts in osteoclast-poor bone, hypoxia, which is temporarily or chronicly oxygen tension lower than the normal bone due to non-smooth blood circulation, Which may lead to deterioration of the bone condition.

Osteoporosis, a typical example of the above-mentioned bone disease, is a disease caused by weakening of bone mineral density due to breakage of equilibrium between bone formation and bone resorption. Today, about 10 million people already suffer from osteoporosis disease in the United States, and 18 million people are at risk of osteoporosis have. In addition, one in two women and one in eight men experience lifelong fractures associated with osteoporosis, and over two million Americans already have osteoporosis. In the United States, direct spending on osteoporosis-related illnesses and fractures is about $ 14 billion annually, and about 400 million people in the country are exposed to or at risk for osteoporosis. This is expected to increase further as the society becomes more aged, and it is expected that social spending and mental and economic expenditure of family members will increase.

In order to treat the above-mentioned bone diseases, it is necessary to control the balance between osteoclasts and osteoblasts. Accordingly, there are a bone resorption inhibitor and a bone formation stimulator. In general, the therapeutic agent for bone diseases such as the bone resorption inhibitor or the bone formation stimulating agent should be administered to a patient for a long period of time, so that it should be low in toxicity. Oral administration is preferable to solve the complication of administration. It is true. On the other hand, studies on osteogenesis stimulants promoting osteogenesis by activating osteoblasts in the bone disease treatment agent have been actively carried out, but strengthening of bone mineral density due to osteogenesis stimulation does not necessarily mean reduction of fracture. On the other hand, Denosumab (Prolia ™, Amgen®), which is currently being used to treat osteoclast activity, has been developed and various drugs have been developed. However, considering that the drug treatment should be continued for a long time, Side effects have not been sufficiently verified, and the exact mechanism of osteoclasts has not yet been elucidated.

Accordingly, the present inventors have made intensive efforts to find a natural product capable of inhibiting the onset or progression of a disease induced by bone loss or a bone loss induced by decreasing osteoclast formation by inhibiting osteoclast formation. As a result, It has been confirmed that one compound has a preventive or therapeutic effect on bone diseases by inhibiting the formation and activity of osteoclast, and the present invention has been completed.

One object of the present invention is to provide a pharmaceutical composition for prevention or treatment of bone diseases comprising Sinomenium acutum extract, a fraction thereof, a compound isolated therefrom, or a pharmaceutically acceptable salt of said compound as an active ingredient .

Another object of the present invention is to provide a method for preventing or treating bone diseases, comprising the step of administering the pharmaceutical composition to a subject in need thereof.

It is still another object of the present invention to provide a health functional food composition for preventing or ameliorating osteoporosis, which comprises a bran extract, fractions thereof, a compound isolated therefrom, or a pharmaceutically acceptable salt thereof.

As one aspect for solving the above problems, the present invention provides a method for preventing or preventing osteoporosis comprising an extract of Sinomenium acutum , a fraction thereof, a compound isolated therefrom, or a pharmaceutically acceptable salt of the compound as an active ingredient, A pharmaceutical composition for therapeutic use is provided.

The present invention is based on the first confirmation that the bran extract has an effect of preventing or treating bone diseases by inhibiting the production and migration of osteoclasts. In particular, four compounds out of the 11 active ingredients extracted from these compounds showed such an effect.

Preferably, the compound isolated from the fragrance extract or its fractions may be salutaridine, dauricumine, cheilanthifoline or dauriporphine.

The term " Sinomenium " acutum "is a deciduous vine plant distributed in Korea, China, Japan, etc. It is 7 m long, has no hair on the branches, has vertical lines, leaves are alternate, round or wide ovate, occasionally shallowly divided into 3 pieces. The flower is bivalent, bloom in June, light green, and hangs on cones. It has 6 calyxes and petals and 9 to 12 stamens, but in female flowers There are 3 hunger and 3 heartbeats, and the nuclei are ripened black in October, and the taste is very high, and the quality is cold.The stem and roots are used for neuralgia and diuretic in one room. It is also used for athletic spasms, paralysis, and Guan-sha. Excretion of water is remarkable, and it is widely used for symptoms such as skin swelling, revenge, It has also been reported to lower blood pressure clinically, to have antitumor effects and to be used as an intravenous injection for the treatment of lung cancer.We have been known to eat, Its preventive or therapeutic effect on bone diseases has not been disclosed to date.

The term "extract of the present invention " as used herein means a product obtained by extracting at least one part of the various parts of the barrier, for example, the stem, roots, leaves, buds and pollen of the barrier. Preferably by extracting stem, root or both together.

The term "extract" of the present invention refers to a preparation which is prepared by squeezing a herbal medicine with an appropriate leaching solution and concentrating by evaporating the leaching solution. The diluent, concentrate or extract of the extract obtained by the extraction treatment is dried A dried product to be obtained, a controlled preparation thereof or a purified product thereof. The fragrant extract may be prepared by a common extraction method, separation and purification method known in the art. The extraction method may be, but not limited to, hot water extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction.

According to a specific embodiment of the present invention, the fragrant extract of the present invention is extracted by adding 10 to 20 times of a solvent to a predetermined weight of dried fragrance. As the solvent, an organic solvent may be used, and the organic solvent may be C _{1 -4} lower alcohol or a mixture thereof, preferably methanol. Further, the extraction can be preferably carried out by a cold extraction method. The cold extraction may be carried out for 3 to 15 days, preferably 5 to 10 days, but is not limited thereto. The extraction can be repeated several times, and the extracted sample is filtered and concentrated under reduced pressure using a vacuum rotary evaporator.

The term "fraction " of the present invention means a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents. The separation of the specific component or the specific group can be achieved by a difference in solubility in these solvents using a concentration gradient of a mixed solvent having a specific property or a mixed solvent containing two or more kinds of solvents, But is not limited thereto. Preferably, the fragrant extract fraction of the present invention may comprise a compound soluble in ethyl acetate, C _{1 -4} lower alcohol or a mixed solvent thereof. More preferably, it may include, but is not limited to, ethyl acetate or n-butanol soluble compounds.

In a specific example of the present invention, the methanol extract of the crude product is sequentially extracted with ethyl acetate and n-butanol to obtain an ethyl acetate soluble fraction and an n-butanol soluble fraction, and the mixture is subjected to column chromatography using a mixed solvent of dichloromethane and methanol Chromatography was performed to obtain several fractions. At this time, a concentration gradient elution system in which the mixing ratio of the dichloromethane / methanol mixed solvent, which is a moving phase, was sequentially increased from 100: 1 to 1: 1 was applied.

Compounds isolated from the fragrance extract or fractions thereof of the present invention can be used in the form of pharmaceutically acceptable salts. Salts are useful as acid addition salts formed by pharmaceutically acceptable free acids. The term "pharmaceutically acceptable salt" of the present invention means a concentration that has a relatively nontoxic and harmless effective action in a patient, and that the side effect caused by the salt is not a deleterious effect of the compound of the present invention Means any organic or inorganic addition salt of the compound. In addition, the compounds of the present invention may be used alone or in combination with other pharmaceutically active compounds or in aggregates.

The pharmaceutically acceptable salt of the compound of the present invention isolated from the fragrance extract or the fractions thereof means a salt prepared according to a conventional method in the art, and such a preparation method is well known to those skilled in the art. In particular, the pharmaceutically acceptable salts include, but are not limited to, salts derived from inorganic and organic acids and bases which are pharmacologically or physiologically acceptable.

The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration. As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycollic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid and the like can be used , But are not limited to these.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal salt or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble salt salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically acceptable to produce sodium, potassium, or calcium salt, but not limited thereto. The corresponding silver salt can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

Pharmaceutically acceptable salts of the compounds separated from the fragrance extract or its fractions include salts of acidic or basic groups which may be present in the compounds isolated from the fragrance extract or fractions thereof, unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of hydroxy groups, and the other pharmaceutically acceptable salts of amino groups include hydrobromides, sulphates, sulphates, phosphates, hydrogen phosphates (Hydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts and the like, ≪ / RTI >

The salt of the compound of the present invention isolated from the fragrance extract or the fractions thereof is not particularly limited as long as it is a pharmacologically acceptable salt and isomeric isomers of the compound derivatives exhibiting osteoclast generation or migration inhibitory effect equivalent to the compound derivatives It is possible.

The present invention also provides a method of preventing or treating bone diseases, comprising the step of administering the pharmaceutical composition to a subject in need thereof.

The composition of the present invention can be used without limitation in bone diseases caused by the presence of more osteoclasts than necessary.

As used herein, the term "bone disease" refers to a condition or disease caused by excessive production and / or migration of osteoclasts, including bone loss diseases. The term "lowering bone disease" refers to a condition or disease in which a decrease in bone mass accompanied by a symptom such as a decrease in bone density and a deterioration of a bone tissue is present. Examples of the disease include osteoporosis, Paget's disease, Periodontal disease, bone growth disorders, and rheumatoid arthritis. Bone loss by osteoclasts activated by cancer cells also falls within the scope of the present invention.

Preferably, the composition of the present invention can be used for the prevention or treatment of osteoporosis or osteopenia. Specifically, the term "osteoporosis" used in the present invention means a condition in which bone mass is decreased and fracture is likely to occur due to a qualitative change, and "osteopenia" means an initial symptom of osteoporosis. In general, osteopenia is classified as -1.0 to -2.5 based on the bone mineral density (T value), and osteoporosis is classified as -2.5 or more.

As used herein, the term "prevention or treatment of bone disease" includes prevention and complete or partial treatment of the bone disease achieved by administering a composition according to the present invention to a subject. It also includes reduction or amelioration of symptoms of bone disease, relief of the symptoms of pain, reduction in the incidence of bone disease, or any other change in the patient that increases the outcome of the treatment.

As used herein, the term "individual" refers to all animals, including humans, who have developed or are capable of developing bone disease. The composition of the present invention can be administered to an individual to effectively prevent or treat the bone disease. The composition of the present invention may be administered in combination with a conventional bone disease therapeutic agent.

As used herein, the term "administering " means introducing a predetermined substance into a patient in an appropriate manner, and the administration route of the composition of the present invention is administered through any conventional route so long as it can reach the target tissue . But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is determined by the patient's sex, Including, but not limited to, medicaments and other medical fields that are used in combination, or in combination with, or in combination with, a pharmaceutically acceptable carrier, excipient, Can be readily determined by those skilled in the art according to known factors. Generally, the active substance may be administered at a dose of from about 0.01 mg / kg / day to 1000 mg / kg / day. When administered orally, a dose of 50 to 500 mg / kg may be appropriate, and may be administered at least once a day.

Preferably, the composition of the present invention is characterized by inhibiting the production or migration of osteoclasts. As used herein, the term "osteoclast" is a large multinuclear cell that engages in bone resorption and is a cell that melts calcified cartilage and bone tissue to allow bone growth. It has 2 to 100 densely packed oval nuclei with a diameter of 20 to 100 μm, and typical osteoclasts are present in shallow, small grooves of the bone surface formed by oneself. The osteoclast located on the bone resorption surface has a bright band and a wave-like edge, and the absorption of the organic substrate by the hydrolytic enzyme secreted by the osteoclast, absorption of the inorganic matter by the acid, and absorption of the bone occur It is known. The activity of osteoblasts forming bones and osteoclasts reabsorbing bones is organically balanced to regulate the amount of bone tissue.

The osteoclasts migrate from the bone to the microfracture site by chemotaxis. It is formed by cell fusion of mononuclear-macrophage cell line and has the characteristic of expressing TRAP (Tartrate Resistant Acid Phosphatase) and cathepsin K at a high level. It is also characterized by cytoplasm with a homogeneous (foamy appearance) appearance due to high concentrations of vesicles and vacuoles. The fear is a lysosomes filled with acid phosphatase. The endoplasmic reticulum of osteoclasts is sparse, and the Golgi complex is extensive. The corrugated border that is useful for the removal of bone-like matrix is a morphological feature of osteoclasts that actively resorb bone. The mineral portion of the substrate, called hydroxyapatite, contains calcium and phosphate ions, which are absorbed into small vesicles by endocytosis and migrate across the cell, resulting in extracellular fluid extracellular fluid to increase the concentration of ions in the blood.

The osteoclast is regulated by parathyroid hormone (PTH) secreted from the parathyroid gland, calcitonin secreted from the thyroid gland, and growth factor interleukin 6 (IL-6). IL-6 is one of the factors of diseases such as osteoporosis due to an imbalance between aggregate absorption and osteogenesis. Also, the activity of osteoclasts is mediated by the interaction of two molecules, osteoprotegerin and RANK ligand, produced by osteoblasts, and osteoclast differentiation is regulated by these two molecules. Excessive production and / or migration of osteoclasts may cause various bone diseases. The fragments of the present invention, fractions thereof, the compounds isolated therefrom, or the pharmaceutically acceptable salts of the compounds or the compounds, Can be treated by inhibiting the production or migration.

In a specific embodiment of the present invention, the antimicrobial extract according to the present invention or a compound isolated therefrom is treated with salutaridine, dauricumine, cheilanthifoline or dauriporphine , It was confirmed that the differentiation into osteoclasts was remarkably inhibited even under culture conditions inducing osteoclast differentiation in which M-CSF and RANKL were added to the culture medium. In addition, since these extracts or compounds do not exhibit cytotoxicity even when treated to a concentration of 10 μg / ml, they can be effectively used as a pharmaceutical composition or a health functional food composition for preventing, ameliorating or treating bone diseases.

Preferably, the pharmaceutical composition of the present invention may contain 0.001 to 1% by weight of the above-mentioned fragrance extract, fractions thereof, a compound isolated therefrom, or a pharmaceutically acceptable salt of the compound or the compound.

In addition, the pharmaceutical composition for preventing or treating bone diseases according to the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-mentioned effective ingredient. As used herein, the term "pharmaceutically acceptable" means that the composition is free of toxicity to an individual such as a cell or human being exposed to the composition. Such carriers may be used without limitation as long as they are known in the art such as buffers, preservatives, wetting agents, solubilizers, isotonic agents, stabilizers, bases, excipients and lubricants. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When the composition of the present invention is formulated, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, and surfactants which are usually used.

For oral administration, solid preparations such as tablets, pills, powders, granules, and capsules can be prepared. Such a solid preparation is prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. in addition to the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agent sweeteners, fragrances and preservatives are included . If the active ingredient is susceptible to degradation by an acid, the oral composition may be formulated to coat the active agent or protect it from degradation from above. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like. Carbohydrates such as glucose, sucrose, and dextran, antioxidants such as ascorbic acid, glutathione, chelating agents, low molecular weight proteins, or other stabilizers may be used to increase stability or absorbency of the active ingredient.

The composition of the present invention may also be administered by any device capable of moving the active substance to the target cell. The preferred modes of administration and formulations are intravenous, subcutaneous, intradermal, intramuscular, and drip injections. The injectable solution may be a non-aqueous solvent such as an aqueous solvent such as a physiological saline solution or a ring gel solution, a vegetable oil, a higher fatty acid ester (e.g., oleic acid), an alcohol (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.) (For example, ascorbic acid, sodium hydrogen sulfite, sodium pyrophosphate, BHA, tocopherol, EDTA and the like), an emulsifier, a buffer for pH control, a microbial growth inhibitor And a pharmaceutical carrier such as a preservative (e.g., mercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

Accordingly, the present invention provides a method for treating a bone disease comprising administering to a subject a pharmaceutical composition comprising the above-mentioned fragrant extract, fractions thereof, a compound isolated therefrom, or a compound or a pharmaceutically acceptable salt of said compound as an active ingredient Of the invention.

In another aspect, the present invention provides a health functional food composition for preventing or ameliorating osteoporosis, which comprises a bran extract, fractions thereof, a compound isolated therefrom, or a pharmaceutically acceptable salt thereof, as an active ingredient.

The composition of the present invention can be used simultaneously or separately with a medicament for the treatment of a disease before or after the onset of osteopathy to prevent or ameliorate a bone disease.

As used herein, the term "improvement" means any action that at least reduces the degree of symptom associated with the condition being treated.

When the health functional food composition of the present invention is used as a food additive, the above-mentioned fragrant extract, its fractions or a compound isolated therefrom may be directly added or used together with other food or food ingredients, . The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use such as prevention, health, or therapeutic treatment. In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, it may be below the above range.

The term "health functional food" used in the present invention refers to a food prepared by processing a specific ingredient as a raw material for the purpose of health assisting or by extracting, concentrating, refining, or mixing a specific ingredient contained in the raw material Refers to foods that are designed and processed so that the body control function such as bio-defense, regulation of biorhythm, prevention and recovery of disease, and the like can be sufficiently exhibited to a living body by the above components. And the like.

There is no limitation on the kind of health food to which the composition of the present invention can be used. In addition, the composition comprising the compound of the present invention as an active ingredient can be prepared by mixing other suitable auxiliary ingredients and known additives, which may be contained in health functional foods, according to the selection of a person skilled in the art. Examples of foods that can be added include dairy products, such as meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Vitamin complex, and the like, and can be prepared by adding to the juice, tea, jelly, and juice prepared from the extract of the present invention as a main component. In addition, it includes all the health foods in the ordinary sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like . The ratio of the natural carbohydrate can be appropriately determined by a person skilled in the art.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The ratios of these additives can also be appropriately selected by those skilled in the art.

The fragrant extract of the present invention, its fractions or the compounds isolated therefrom are used as a raw material for natural herbal medicine, so that they can be less harmful than general synthetic compounds even when they are used as pharmaceutical compositions or food additives, And can be usefully used.

The extract of Sinomenium acutum according to the present invention, its fractions and the compounds isolated therefrom, especially salutaridine, dauricumine, cheilanthifoline and / or dauriporphine, Or a pharmaceutically acceptable salt of said compound as an active ingredient inhibits osteoclastosis due to bone loss by inhibiting the differentiation and activity of osteoclast, Can be usefully used.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. (A) shows a microscope image for TRAP staining, (b) shows TRAP activity, and (c) shows survival rate for the cells.
2 is a diagram showing the structural formulas and names of the active ingredients (Compounds 1 to 11) isolated from the fragrance extract according to the present invention.
FIG. 3 is a graph showing the effect of RANKL-induced osteoclast formation according to the treatment of Compounds 1 to 4, 8, 10, and 11 isolated from the fragrance extract according to the present invention. (A) shows a microscope image for TRAP staining and (b) shows TRAP activity for the cells cultured and treated with the above compound in concentration.
FIG. 4 is a graph showing RANKL-induced osteoclast formation inhibitory effects of Compounds 5, 8, 10, and 11, which were isolated from the fragrance extract of the present invention. (B) shows the number of TRAP-positive polynucleated osteoclasts (MNC), (c) shows the number of TRAP-positive cells in the cells . ≪ / RTI >

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: Bangui  Extract and its Fraction  Produce

Dried bran ( Sinomenium acutum ) 6 kg were weighed, placed in an extraction container, and 90 L of methanol was added thereto. The mixture was then cooled by freezing for 7 days and filtered. The obtained extract was concentrated under reduced pressure using a vacuum rotary evaporator. The above extraction and concentration process was repeated twice to obtain 737 g of a crude extract.

The resultant fragrant methanol extract was suspended in 4 L of distilled water, adjusted to pH 12.0 with 1N NaOH, and extracted with an equal volume of ethyl acetate. The extract obtained by repeating the extraction process three times was combined and concentrated under reduced pressure to obtain 84 g of ethyl acetate fraction. The filtrate obtained from the extraction process was repeatedly extracted three times with the same amount of n-butanol, and the extract was concentrated under reduced pressure to obtain 72 g of a butanol fraction.

Example  2: Bangui  Isolation and Identification of Active Ingredients from Extracts

Eleven kinds of compounds as active ingredients were isolated from the fragrant extract prepared in Example 1, and the structural formula and the names of these compounds are shown in Fig.

Specifically, the ethyl acetate fraction (84 g) and the n-butanol extract (72 g) of the fragrant methanol extract obtained from Example 1 were named as an alkaloid fraction (156 g). In addition, in the ethyl acetate fraction, 5.5 g of synomenine (Formula 1 in Fig. 2) was separated into a crystalline state. 150.5 g of the alkaloid fraction except for cinomenine was flowed into a mobile phase with a dichloromethane / methanol mixed solution, and the concentration ratio of dichloromethane / methanol was sequentially increased from 100: 1 to 1: 1, Chromatography was performed to obtain a total of five fractions (fractions 1 to 5).

The obtained first fraction was subjected to amine gel column chromatography (mobile phase: n-hexane / ethyl acetate = 10: 1) to separate 3.1 mg of dauripopine (Formula 11 of FIG. 2). The second fraction was subjected to reverse phase column chromatography (magic: river space carbon-18, mobile phase: 30 to 90% methanol) to obtain 37.1 mg of 8-demethoxyaraninine (formula 4 in FIG. 2) (Formula 10 in Fig. 2) of 6.0 mg. The third fraction was subjected to reverse phase column chromatography under the same conditions as in the second fraction to obtain 23.8 mg of 14-epithinomenine (Formula 2 in Fig. 2), 37.2 mg of salutaridine (Formula 5 in Fig. 2) 58.7 mg of acutumine (Formula 6 of FIG. 2), 7.4 mg of acutumidine (Formula 7 of FIG. 2), and 9.3 mg of dauricumin (Formula 8 of FIG. In addition, the fourth fraction was subjected to amine gel column chromatography (mobile phase: n-hexane / ethyl acetate = 5: 1) to separate 92.4 mg of synomene N-oxide (Formula 3 in FIG. 2) 2.1 g of magnesium fluorine (Formula 9 in Fig. 2) was isolated.

The separated compounds were identified by ¹ H-NMR and ¹³ C-NMR.

Example  3: Bangui  Evaluation of inhibitory effect on osteoclast formation of extracts and compounds isolated therefrom

Five-week-old male ICR mice were purchased and the femur and tibia were taken and washed with α-MEM containing antibiotics (100 units / ml penicillin and 100 μg / ml streptomycin) to form bone marrow cells ). The bone marrow cells were cultured in α-MEM containing 10% fetal bovine serum (FBS) and M-CSF (macrophage colony-stimulating factor; 10 ng / ml) for 1 day. Non-adherent bone marrow cells were seeded in Petri dishes and cultured for 3 days in the presence of M-CSF (30 ng / ml). Adherent cells were used as bone marrow-derived macrophages (BMM) after washing nonadherent cells. BMM cells were treated with DMSO or the bran extract prepared in Example 1 (1, 3 and 10 μg / ml) in the presence of RANKL (10 ng / ml) and M-CSF Lt; / RTI > Cells treated with M-CSF without RANKL were used as negative controls. Multinucleated osteoclasts were fixed with 3.7% formalin for 10 minutes and treated with 0.1% Triton X-100 for 10 minutes to have permeability. Cells were stained with TRAP solution (Sigma-Aldrich). Multinucleated osteoclasts were visualized by TRAP (tartrate resistant acid phosphatase) staining (Fig. 1A). The TRAP activity was determined according to the concentration of the extract, and FIG. 1b shows that the TRAP activity was effectively reduced to 30 ~ 40% when 10 .mu.g / ml of the extract was treated. The cell viability was calculated in the cells treated with the same concentration of the phytotoxic extract in order to confirm whether the phytotoxic extract had cytotoxicity and is shown in FIG. 1C. As shown in FIG. 1C, it was confirmed that the cell survival rate was increased in the cell group treated with 1 ~ 10 μg / ml of the extract of Bombyx mori than in the control group without Bombyx mori extract. This indicates that the bran extract does not show cytotoxicity within the concentration range.

The 11 compounds (FIG. 2) isolated from Example 2 above were each treated at different concentrations and cultured. The cells were cultured in the same manner as the cells cultured and treated with the repellent extract to perform TRAP staining. The results for the compounds (compounds 1 to 4, 6, 7 and 9) of which the effect of inhibiting TRAP activity is insignificant among the above 11 compounds are shown in Fig. 3, and the compounds (compounds 5, 8 and 10 And 11) are shown in Fig.

As shown in FIG. 3, the compounds 1 to 4, 6, 7, and 9 exhibited TRAP activity at a level similar to that of the non-treated group, even at a treatment concentration of 3 μg / ml, It was confirmed that it did not show the effect.

Compounds 5, 8, 10, and 11, on the other hand, exhibited remarkable TRAP activity inhibitory effects (Fig. 4A), which can be confirmed visually even at a concentration of 3 μg / ml. Of these cells, TRAP-expressing TRAP-positive multinucleated osteoclast cells (MNCs) and multicellular cells having three or more nuclei were counted separately and shown in FIG. 4b, and the compound of the present invention Of osteoclast formation was evaluated. As is apparent from the naked eye, the number of MNCs began to decrease rapidly from the experimental group treated with 3 μg / ml concentration, and almost no MNC appeared in all four compounds in the experimental group treated with 10 g / ml concentration. Furthermore, the compounds showing the TRAP activity inhibitory effect were also measured for cell viability to determine whether they exhibited cytotoxicity, and the results are shown in FIG. 4C. As shown in FIG. 4C, the survival rate of 100% or more was observed up to 5 μg / ml for all four compounds. The cell survival rate was slightly decreased in the cells treated with 10 μg / ml of the compound 10 and 11, %, And compounds 5 and 8 showed a survival rate of 100% or more even at a concentration of 10 μg / ml.

Claims (8)

A pharmaceutical composition for the prevention or treatment of osteoporosis comprising, as an active ingredient, the following compounds, their pharmaceutically acceptable salts, or a mixture thereof, isolated from a Sinomenium acutum extract or a fraction thereof:

(Salutaridine),

(Dauricumine),

(Cheilanthifoline),

(Dauriporphine).
delete delete delete The method according to claim 1,
Wherein the composition inhibits osteoclast formation or migration.
The method according to claim 1,
Wherein the bone disease is selected from the group consisting of osteoporosis, Paget's disease, periodontal disease, bone-growth disorder, and rheumatoid arthritis.
The method according to claim 1,
A pharmaceutical composition containing the compound or a pharmaceutically acceptable salt of the compound in an amount of 0.001 to 1% by weight.
A health functional food composition for preventing or ameliorating a bone disease comprising, as an active ingredient, the following compound, a pharmaceutically acceptable salt thereof, or a mixture thereof:

(Salutaridine),

(Dauricumine),

(Cheilanthifoline),

(Dauriporphine).

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