KR101809151B1 - Composition for prevention, improvement or treatment of osteoporosis comprising kirenol or extract of Sigesbeckia spp. - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating osteoporosis comprising kirenol or an extract of Hui Chum ( Siegesbeckia spp.) Containing the same as an active ingredient. The kirenol according to the present invention, or the extract of the present invention containing the same or a fraction thereof, has an excellent effect for preventing, improving or treating osteoporosis as it promotes osteoblast differentiation and inhibits osteoclastogenesis. In addition, since the present invention is a natural product, it can be safely used without side effects and can be used as a medicine or food.

Description

[0001] The present invention relates to a composition for prevention, improvement or treatment of osteoporosis, comprising a quinolone or a quinolone extract.

The present invention relates to a composition for preventing, ameliorating or treating osteoporosis comprising kirenol or Sigesbeckia spp. Extract as an active ingredient.

Osteoporosis is the most common skeletal disease among bone related diseases such as multiple myelosis and osteoarthritis, and is generally characterized by an increase in fracture and a decrease in bone strength. For normal bone function, bone remodeling by bone homeostasis of bone formation by osteoclast and osteoblast is needed (for example, Nat. Rev. Endocrinol. 8: 212-227, 2011, J. Dent. Res. 91: 736-744, 2012).

However, excessive activity of osteoclasts and deactivation of osteoblasts cause imbalance in the remodeling process, leading to adult skeletal diseases such as osteoporosis. In order to alleviate this imbalance, methods for suppressing excessive activity of osteoclasts, promoting osteoclast activity, inhibiting osteoclast activity and promoting osteoclast activity are generally used, and they are used for treatment of osteoporosis (Nat. Rev. Rheumatol. 7: 631-638, 2011).

Osteoblasts are formed from mesenchymal stem cells. The mineralization, including calcium, formed 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, and it is known that bone morphogenetic protein (BMP), Wnt, MAP kinase, calcineurin-calmodulin kinase Alkaline phosphatase (ALP), which is involved in osteoblast differentiation by cross-talk of various signaling systems such as calcineurin-calmodulin kinase, NF-κB and AP-1, (Osteopontin), osteocalcin (OCN), type I collagen (Type I collagen; ColA1), etc. associated with mineralization are known to be involved in bone formation by osteoblast differentiation Nat. Rev. Drug Discov. 11: 234-250, 2012).

The Wnt / β-catenin signal transduction system is a co-receptor of the signal transduction system, LRP5 (low-density lipoprotein-receptor-related protein-5), osteoporosis pseudoglioma syndrome (OPPG) Has been actively studied for bone growth and bone density regulation since it is reported to be a cause of bone resorption. It is known that activation of the Wnt / beta -catenin signaling system induces an increase in bone volume, and effective and safe treatment of osteoporosis (Cell Signal. 20: 999-1009, 2008, Nat. Med. 19: 179-192, 2013, Sci. Transl. Med. 28: 36-42, 2010, Curr. Mol. Pharmacol 4: 14-25 2011).

A representative treatment for osteoporosis developed so far is bisphosphonates, which inhibits bone loss by weakening the functions of osteoclasts (bone resorption-related cells), and Merck's Fosamax, Procter Gamble And "Actonel" of the company. However, these treatments do not promote the formation of bones, and as a decomposition-inhibiting agent, it is necessary to substitute bone because it can induce additional fractures in continuous use, so it can not be a fundamental treatment. (Datamonitor Research Reports, 2007). Therefore, the present inventor has conducted research to develop a raw material for prevention and treatment of osteoporosis using natural materials having a low safety, such as plant extracts, which are not chemical synthetic products.

Cell Signal. 20: 999-1009, 2008 Nat. Rev. Drug Discov. 11: 234-250, 2012

The present inventors have found that the results of studies in order to search for a natural substance which can be applied has the activity enhancing effect of bone cells safely, Kastrup play (kirenol) or huicheom (Hui Chum, Siegesbeckia spp.) Extracts or fractions thereof containing the same osteoporosis Prevention, amelioration or therapeutic effect of the present invention.

As means for solving the above problems,

A pharmaceutical composition for preventing or treating osteoporosis comprising an extract of Orissa or a fraction thereof as an active ingredient, and a food composition for preventing or improving osteoporosis.

As another means for solving the above problems,

A pharmaceutical composition for preventing or treating osteoporosis and a food composition for preventing or improving osteoporosis, which comprises a compound represented by the following formula (1) as an active ingredient:

[Chemical Formula 1]

The kirenol according to the present invention, or the extract of the present invention containing the same or a fraction thereof, has an excellent effect for preventing, improving or treating osteoporosis as it promotes osteoblast differentiation and inhibits osteoclastogenesis. In addition, since the present invention is a natural product, it can be safely used without side effects and can be used as a medicine or food.

FIG. 1 shows the results of measuring mRNA expression levels (left) and relative mRNA expression levels (right) of osteoblast differentiation genes (ALP, ColA1 and OPN) in MC3T3-E1 osteoblast by treatment with pyrenol.
FIG. 2 shows the results of measuring the activity of alkaline phosphatase (ALP), which is an early differentiation marker of osteoblast-induced osteoclast-induced osteoblast-like cells in MC3T3-E1 osteoblasts.
FIG. 3 shows the results of measuring the effect of osteoblast mineralization enhancement by the treatment with chelolnol on MC3T3-E1 osteoblast cells.
FIG. 4 shows the mRNA expression level (left) of osteoclast production inhibitory gene (OPG (osteoprotegerin), receptor activator of nuclear factor kappa-B ligand (RANKL)) (left side) and mRNA relative to the control group in MC3T3-E1 osteoblast And the expression level (right).
FIG. 5 shows the mRNA expression levels (left) and relative mRNA expression levels of the bone morphogenic protein (BMP) signal transduction system genes (BMP2, Runx2, and Osx) in the MC3T3-E1 osteoblast Right).
FIG. 6 shows the mRNA expression levels (left) and relative mRNA expression levels of the Wnt /? -Catenin signaling system genes (LRP5, DVL2,? -Catenin and CCND1) in the MC3T3-E1 osteoblasts ), Respectively.
FIG. 7 is a graph showing changes in osteoblast differentiation genes (ALP, ColA1, OCN, and the like) in MC3T3-E1 osteoblast by treatment with Shigeysbeckia aggrabresens ethanol extract, Shigesbeckia afubensense ethanol extract and Shigesbeckia orientalis ethanol extract OPN) mRNA expression levels of the cells.
FIG. 8 is a graph showing the results of inhibition of osteoclastogenesis (OPG and RANKL) by osteoblastic cells of MC3T3-E1 osteoblasts, ethanol extract of Shigesbeckia glabensense, ethanol extract of Shigesbeckia fulvensens and ethanol extract of Shigesbeckia orientalis ) MRNA expression levels of the cells.
9 shows mRNA expression levels of osteoblast differentiation genes (ALP and ColA1) by treatment with methanol extract, hot water extract and ethyl acetate extract of Shigesbeckia orientalis in MC3T3-E1 osteoblast.
10 shows mRNA expression levels (left) and relative mRNA expression levels (right) of osteoblast differentiation genes (ALP, ColA1, OCN, and OPN) in MC3T3-E1 osteoblast treated with Shigesbeckia orientalis extract, The results are shown in Fig.
FIG. 11 shows the results of measuring the activity of alkaline phosphatase (ALP) as an early differentiation marker of osteoblast by treatment with Shigesbeckia orientalis extract at MC3T3-E1 osteoblast.
FIG. 12 shows the results of measurement of the osteoblast mineralization enhancement effect of MC3T3-E1 osteoblast by treatment with Shigesbeckia orientalis extract.
FIG. 13 shows mRNA expression levels (left) and relative mRNA expression levels (right) relative to the control group in osteoclast cells (OPG and RANKL) inhibiting osteoclast formation by treatment with Shigesbeckia orientalis extracts in MC3T3-E1 osteoblasts The results of the measurement are shown.
14 shows the mRNA expression levels (left) and relative mRNA expression levels of the osteogenic protein signaling pathway gene (BMP2, Runx2, and Osx) in the MC3T3-E1 osteoblast by treatment with Shigesbeckia orientalis extract ), Respectively.
15 shows the mRNA expression levels (left) of the Wnt /? -Catenin signaling system genes (LRP5, DVL2,? -Catenin, and CCND1) in MC3T3-E1 osteoblast cells treated with Shigesbeckia orientalis extract And the relative mRNA expression level (right).

Hereinafter, the configuration of the present invention will be described in detail.

The present invention provides a method for preventing, ameliorating or treating osteoporosis of a pleiotropic extract or a fraction thereof, or a compound represented by the following formula (1): A composition for preventing, ameliorating or treating osteoporosis, comprising an extract according to claim 1 or a fraction thereof, or a compound represented by the following formula (1); Or fragments thereof, or a compound represented by the following formula (1), to a mammal including a human.

[Chemical Formula 1]

In this specification "huicheom" or "huiryeom 'is's Shigeru of Asteraceae plants Becky subgenus (Siegesbeckia spp.) Siegesbeckia glabrescens Mak.), Siegesbeckia pubescens Mak.) or Siegesbeckia orientalis L.). Shigesbukki Agla Bresens is a courtesan, Shiges Becky Aphubesense is a courtesan, and Shigesbeckia Orientalis is a Jeju court.

In the present specification, the 'excipient extract' or the 'comedium extract' is used interchangeably and means an extract obtained by extracting excipients (diluents). The method for preparing the excipient extract can be carried out by any conventional extraction method known in the art without limitation, for example, from water (plant or plant part) (leaf or root), water, an organic solvent having 1 to 6 carbon atoms, Or a supercritical fluid, and extracting it with at least one solvent selected from the group consisting of a supercritical fluid.

As used herein, the term " fraction " means a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents. Specific fractions enriched in active material can be prepared using techniques such as solvent fractionation, silica gel chromatography, prep-HPLC, and the like.

The term " improvement " or " treatment " means alleviating, alleviating, or eliminating the symptoms of osteoporosis, delaying the progress of osteoporosis, Or reversing, such as osteoporosis. The term "composition for preventing, improving or treating osteoporosis" includes a pharmaceutical composition or a food composition.

The composition for preventing, ameliorating or treating osteoporosis of the present invention may further contain one or more active ingredients showing the same or similar functions in addition to the excipient extract or its fractions. For example, it may contain known components effective for osteoporosis. The inclusion of additional ingredients may further enhance the prevention, amelioration or therapeutic effect of the composition of the present invention on osteoporosis. When the above ingredients are added, skin safety, easiness of formulation, and stability of effective ingredients can be considered according to the combined use. In one embodiment of the present invention, the composition comprises an extract of Gardeniae Fructus , Crinumasiaticum extract, silkworm powder, rhizome extract, Rhus verniciflua Stokes extract, Extracts of Ganoderma lucidum, extracts of Ganoderma lucidum, extracts of Ganoderma lucidum, extracts of Ganoderma lucidum, extracts of Ganoderma lucidum, extracts of Ganoderma lucidum, extracts of Ganoderma lucidum, extracts of Ganoderma lucidum, Mint extract, ginger extract, and the like may be further included. The additional component may be contained in an amount of 0.0001 wt% to 10 wt% based on the total weight of the composition. For example, from 0.0001 wt% to 1 wt%, from 0.0001 wt% to 0.1 wt%, from 0.0001 wt% to 0.001 wt%, from 0.001 wt% to 10 wt%, from 0.001 wt% 0.001 wt% or more to 0.1 wt% or less, 0.01 wt% or more to 10 wt% or less, or 0.01 wt% or more to 1 wt% or less. The content range may be adjusted according to requirements such as skin safety, ease of formulation of the compound of Formula 1, and the like.

The present invention provides a pharmaceutical composition for preventing or treating osteoporosis or a food composition for preventing or ameliorating osteoporosis, wherein the composition comprises a chelate extract or a fraction thereof as an active ingredient.

In one embodiment, the excreted extract may be an extract of one or more plants selected from the group consisting of Shigesbeckia glabresense, Shigesbeckia afubesense, and Shigesbeckia orientalis. For example, ethanol extracts, hot-water extracts, hexane extracts, ethyl acetate extracts, ultra-high-pressure extracts using dried leaves and stalks of Shigesbeckia aglabreensense, Shigesbeckia afubensense or Shigesbeckia orientalis have.

In one embodiment, the excipient extract may be obtained by extracting the excipient with one or more solvents selected from the group consisting of water, organic solvents of 1 to 6 carbon atoms, subcritical fluids, and supercritical fluids. For example, it can be obtained by extracting a scarlet plant under an ultra-high pressure condition of 100 MPa or more. If necessary, it can be prepared by further adding filtration and concentration steps according to methods known in the art.

In one embodiment, the organic solvent having 1 to 6 carbon atoms is selected from the group consisting of alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, And may be at least one selected from the group consisting of methylene chloride, hexane, cyclohexane, and petroleum ether.

The excipient extract of the present invention can be obtained by extracting and purifying the dried excipient using purified water, ethanol and subcritical carbon dioxide suitable for food processing, or extracting and purifying it using an ultra-high pressure extraction apparatus , Or by separating and purifying the oil obtained by directly pressing the scarlet plant. For example, the extract can be obtained by extracting the extract under an ultra-high pressure condition of 100 MPa or more.

In one embodiment, the scavenger extract or fraction thereof may contain a keyenol as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention or treatment of osteoporosis comprising the compound represented by the formula (1) as an active ingredient, or a food composition for preventing or improving osteoporosis.

[Chemical Formula 1]

The compound of Formula 1 may include all possible isomers, for example, a compound of Formula 2 below.

(2)

The compound of formula (2) is called kirenol.

Kyrenia play (kirenol) is Shige's Becky Ah Oriental lease (Siegesbeckia orientalis L.), Siegesbeckia pubescens Mak.), Siegesbeckia glabrescens Mak.), which belongs to the genus Siegesbeckia inhabiting Jeju and southern regions spp.) is an active ingredient in plants. Chironol or squalane extracts have been shown to have anti-inflammatory and analgesic effects (J. Ethnopharmacol. 137: 1089-1094, 2011), antimicrobial effects (Pharmacogn. Mag. 8: 149-155, 2012), arthritis effects (Phytomedicine 19: 882-889 , 2012), but the effect of preventing and treating osteoporosis has not been known prior to the present invention.

Kirenol can be obtained by extracting and purifying dried excipients using purified water suitable for food processing, ethanol and supercritical carbon dioxide, or extracting and purifying them using an ultra-high pressure extraction device, Oil, and the like. The separation and purification of the quinolone from the excipient extract can be carried out by using column chromatography or high performance liquid chromatography (HPLC) packed with various synthetic resins such as silica gel or activated alumina, alone or in combination, The method of extracting and separating and purifying the quinolone is not necessarily limited to the above method.

The compound of formula (1) or the compound of formula (2) may be isolated from or synthesized from plant extracts, or may be a commercially available compound.

In one embodiment, the compound represented by Formula (1) or the compound represented by Formula (2) may be isolated from a scion extract or a fraction thereof.

The pharmaceutical composition for the prevention or treatment of osteoporosis of the present invention may comprise a pharmaceutically acceptable salt of the choline. The term " pharmaceutically acceptable " as used herein refers to those which are physiologically acceptable and do not normally cause an allergic reaction or a similar reaction when administered to humans, and the salts include pharmaceutically acceptable free acids acid is preferred.

The pharmaceutically acceptable salt of the above-mentioned choline may be an acid addition salt formed using an organic acid or an inorganic acid, and examples of the organic acid include formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, There may be mentioned acetic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, , p-toluenesulfonic acid or methanesulfonic acid. The inorganic acid includes, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid or boric acid. The acid addition salt may preferably be in the form of a hydrochloride or an acetate, more preferably in the form of a hydrochloride.

The above-mentioned acid addition salts may be prepared by a) directly mixing the kinerol and the acid, b) dissolving and mixing one of them in a solvent or a water solvent, or c) placing the kinerol in the solvent or an acid And mixing them.

Separately, additionally saltable forms include, but are not limited to, the salts of gabapentin, gabapentin, pregabalin, nicotinate, adipate, hemimarate, cysteine, acetylcysteine, methionine, arginine, lysine, Aspartate and the like.

In addition, the pharmaceutical composition for preventing or treating osteoporosis of the present invention may further comprise a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. The carrier for parenteral administration may also contain water, suitable oils, saline, aqueous glucose and glycols and the like. In addition, stabilizers and preservatives may be further included. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally, and parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, , Intranasal, enteral, topical, sublingual or rectal administration.

The pharmaceutical composition of the present invention may be formulated into oral or parenteral dosage forms according to the route of administration as described above. When formulated, one or more buffers (e. G., Saline or PBS), carbohydrates (e. G., Glucose, mannose, sucrose or dextran), antioxidants, bacteriostats, chelating agents Diluents or excipients such as suspending agents, diluents or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxides), suspending agents, thickening agents, disintegrating or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, solutions, gels, syrups, slurries, suspensions or capsules, etc. These solid preparations can be prepared by incorporating into the pharmaceutical composition of the present invention at least one excipient, , Starch (including corn starch, wheat starch, rice starch and potato starch), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, , Methyl cellulose, sodium carboxymethyl cellulose and hydroxypropylmethyl-cellulose or gelatin. For example, tablets or tablets may be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and processing the mixture into granules.

In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions or syrups. In addition to water or liquid paraffin, which is a simple diluent commonly used, various excipients such as wetting agents, sweeteners, fragrances or preservatives may be included .

In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant, and may further include an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent .

For parenteral administration, the pharmaceutical compositions of the present invention may be formulated in accordance with methods known in the art in the form of injectable, transdermal and nasal inhalers, together with suitable non-oral carriers. In the case of such injections, they must be sterilized and protected against contamination of microorganisms such as bacteria and fungi. Examples of suitable carriers for injectables include, but are not limited to, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and / or vegetable oils . More preferably, suitable carriers include isotonic solutions such as Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose Etc. may be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like may be further included. In addition, the injections may in most cases additionally include isotonic agents, such as sugars or sodium chloride.

Examples of transdermal dosage forms include ointments, creams, lotions, gels, solutions for external use, pastes, liniments, and air lozenges. By " transdermal administration " as used herein, it is meant that the pharmaceutical composition is locally administered to the skin, whereby an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.

In the case of inhalation dosage forms, the compounds used according to the present invention may be formulated into a pressurized pack or a pressurized pack using a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated to contain a compound, and a powder mixture of a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, commonly known in all pharmaceutical chemistries.

The pharmaceutical composition of the present invention can provide a desired osteoporosis prevention, improvement or therapeutic effect when it contains an effective amount of quinolone or a chelate extract or a fraction thereof. As used herein, the term " effective amount " refers to an amount that exhibits a higher level of response than a negative control, and preferably refers to an amount sufficient to prevent, ameliorate, or treat osteoporosis. The pharmaceutical composition of the present invention may contain 0.01 to 99.99% of pyrenol or a squalane extract containing the same, and the remaining amount may be occupied by a pharmaceutically acceptable carrier. The effective amount of the quinolone or squalane extract or fraction thereof contained in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is produced.

The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses . The pharmaceutical composition of the present invention may vary in the content of the active ingredient depending on the degree of the disease. For example, it can be administered in one to several divided doses so as to be administered in an amount of preferably 0.001 to 100 mg, more preferably 0.01 to 10 mg per kilogram of body weight per day of quinolone or a squalene extract containing the same. However, the dose of the above-mentioned quinolone or the excipient extract containing the same is not limited to the administration route and the number of treatments of the pharmaceutical composition, but may be varied depending on various factors such as the patient's age, body weight, health condition, sex, severity of disease, A person skilled in the art will be able to determine the appropriate effective dose according to the specific use for prevention or treatment of osteoporosis, It will be possible. The pharmaceutical composition according to the present invention is not particularly limited to the formulation, administration route and administration method as long as the effect of the present invention is exhibited.

The pharmaceutical composition for preventing or treating osteoporosis of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy or biological response modifiers.

The pharmaceutical composition for the prevention or treatment of osteoporosis of the present invention can also be provided as a formulation of external preparations containing an active ingredient such as quinolone or a quisaded extract or a fraction thereof.

When the pharmaceutical composition for the prevention or treatment of osteoporosis of the present invention is used as an external preparation for skin, it may further contain a fatty substance, an organic solvent, a solubilizer, a thickening agent and a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, Surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic active agents, Such as lipid vesicles, and any other ingredients conventionally used in dermatologic external preparations. The components can also be introduced in amounts commonly used in the field of dermatology.

When the pharmaceutical composition for preventing or treating osteoporosis of the present invention is provided as an external preparation for skin, it may be a formulation such as an ointment, a patch, a gel, a cream or a spray.

The food composition for preventing or improving osteoporosis of the present invention includes all forms of functional food, nutritional supplement, health food, food additives and feed, Animals including livestock are targeted for eating. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art.

Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art. Common foods include but are not limited to beverages (including alcoholic beverages), fruits and processed foods (eg, canned fruits, jam, maamalade, etc.), fish, meat and processed foods (Eg butter, chewing), edible vegetable oil, margarine (such as corn oil, etc.), breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), juice, various drinks, cookies, , Vegetable protein, retort food, frozen food, various kinds of seasoning (for example, soybean paste, soy sauce, sauce, etc.), or the excipient extract containing the above-mentioned pyrenol. The nutritional supplement is not limited thereto, but it can be prepared by adding the above-mentioned pyrenol or an excreting extract containing the same to capsules, tablets, rings and the like. The health functional foods include, but are not limited to, for example, lanolin, granules, capsules, and the like, which are prepared in the form of tea, juice, and drink, And powdered. In addition, in order to use the above-mentioned quinolone or the excipient extract containing it as a food additive, it may be used in the form of a powder or a concentrate. In addition, it can be prepared in the form of a composition by mixing the above-mentioned quinolone or the excipient extract containing it with a known active ingredient known to be effective for osteoporosis.

When the food composition for preventing or ameliorating osteoporosis of the present invention is used as a health beverage composition, the health beverage composition may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose; Polysaccharides such as dextrin, cyclodextrin; Xylitol, sorbitol, erythritol, and the like. Sweeteners include natural sweeteners such as tau Martin and stevia extract; Synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

The quinolone or the squalane extract containing the quinolone may be contained as an active ingredient of a food composition for preventing or ameliorating osteoporosis, and the amount thereof is not particularly limited in terms of an effective amount for obtaining osteoporosis prevention and improvement effects, It is preferably 0.01 to 100% by weight. The food composition of the present invention may be prepared by mixing together with other active ingredients which are known to be effective in osteoporosis together with choline or a sucrose extract containing it.

In addition to the above, the health food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acid, protective colloid thickener, pH adjusting agent, Glycerin, an alcohol or a carbonating agent, and the like. In addition, the health food of the present invention may contain natural fruit juice, fruit juice drink, or pulp for the production of vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail with reference to examples. It is to be understood, however, that the following examples are provided for illustrative purposes only, and are not intended to limit the scope of the present invention.

Reference Example  One: Kirenol material  Information

Name: Kirenol; Kirel; 3-hydroxy-7 - [(R) -piperazin-1-yl) -1,2-dihydroxyethyl] -1,4a, 7-trimethylphenanthrene-1-methanol

CAS No .: 52659-56-0

[ Example  One] Shigesbekkia Glavensense  Preparation of extract

[Example 1-1] Preparation of ethanol extract of Shigeysbeckia aglabrasense

The leaves and stems of the dried Shigesbeckia aglabresens were pulverized with a mixer, and 100 g of the ground Shigesbeckia glabresense sample was added to 1 L of ethanol and extracted with stirring at 50 DEG C for 60 minutes. The extracted sample was filtered with filter paper of Whatman No. 2, and the filtered extract was concentrated with a vacuum rotary condenser to remove the solvent component to obtain an extract of Shigesbeckia glabensense ethanol.

[Example 1-2] Preparation of methanol extract of Shigeysbeckia aggla brecesense

The leaves and stems of the dried Shigesbeckia aglabresens were pulverized with a mixer, and 100 g of the ground Shigesbeckia glabresense sample was added to 1 L of ethanol and extracted with stirring at 50 DEG C for 60 minutes. The extracted sample was filtered through filter paper of Whatman No. 2, and the filtered extract was concentrated by a vacuum rotary condenser to remove solvent components to obtain a methanol extract of Shigesbeckia glabresense.

[Example 1-3] Preparation of hot-water extract of Shigeysbeckia aggla brecesense

The leaves and stems of the dried Shigesbeckia aglabresens were pulverized with a mixer, and 100 g of the pulverized Shigesbeckia glabresense sample was added to 1 L of water and extracted with stirring at 100 ° C for 4 hours. The extracted sample was filtered with filter paper No. 2 of Wattman, and the filtered extract was concentrated with a vacuum rotary condenser to remove the solvent component to obtain a Shigesbeckia glabresense hot water extract.

[Example 1-4] Preparation of an extract of Shigesbeckia aglabactense ethyl acetate

The leaves and stems of the dried Shigesbeckia aglabresens were pulverized with a mixer, and 100 g of the ground Shigesbeckia glabresense sample was added to 1 L of ethyl acetate and extracted with stirring at 50 DEG C for 60 minutes. The extracted sample was filtered with a filter paper No. 2 of Wattman, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components to obtain an extract of Shigesbeckia glabensense ethyl acetate.

[Example 1-5] Preparation of Extra-high Pressure Extract of Shigeysbeckia aggla brecesense

The leaves and stems of the dried Shigesbeckia aglabresens were pulverized with a mixer, and then 1 g of the Shigesbeckia aggrabresense sample and 76 ml of 18% ethanol were put in a polyethylene pack and sealed. Then, And extracted using an extraction device (Frescal MFP-7000; Mitsubishi Heavy Industries, Tokyo, Japan). Extraction pressure was 320 MPa and extraction time was 5 min. The extracted sample was filtered with filter paper No. 2 of Wattman, and the filtered extract was concentrated with a vacuum rotary condenser to remove the solvent component to obtain a Shigesbeckia glabresense ultrahigh pressure extract.

[ Example  2] Shigesbekkia Fubesense  Preparation of extract

[Example 2-1] Preparation of ethanol extract of Shigesbeckia afubensense

The leaves and stems of the dried Shigesbeckia aglabresens were pulverized with a mixer, and 100 g of the ground Shigesbeckia glabresense sample was added to 1 L of ethanol and extracted with stirring at 50 DEG C for 60 minutes. The extracted sample was filtered through filter paper of Whatman No. 2, and the filtered extract was concentrated with a vacuum rotary condenser to remove the solvent component to obtain an extract of Shigesbeckia fubesense ethanol.

[Example 2-2] Preparation of methanol extract of Shigesbeckia afubesense

The leaves and stems of the dried Shigesbeckia aglabresens were pulverized with a mixer, and 100 g of the ground Shigesbeckia glabresense sample was added to 1 L of ethanol and extracted with stirring at 50 DEG C for 60 minutes. The extracted sample was filtered through filter paper of Whatman No. 2, and the filtered extract was concentrated by a vacuum rotary condenser to remove solvent components to obtain a methanol extract of Shigesbacquiapubesense.

[Example 2-3] Preparation of hot water extract of Shigesbeckia afubesense

The leaves and stems of the dried Shigesbeckia abbenseens were pulverized with a mixer, and then 100 g of the pulverized Shigesbeckia fubesense sample was added to 1 L of water and extracted with stirring at 100 ° C for 4 hours. The extracted sample was filtered with filter paper No. 2 of Wattman, and the filtered extract was concentrated with a vacuum rotary condenser to remove the solvent component to obtain a Shigesbeckia fubesense hot water extract.

[Example 2-4] Preparation of an extract of Shigeysbeckia afubesense ethyl acetate

The leaves and stem of the dried Shigesbeckia afubensense were pulverized with a mixer, and 100 g of the pulverized Shigesbeckia fubesense sample was added to 1 L of ethyl acetate and extracted with stirring at 50 ° C for 60 minutes. The extracted sample was filtered with filter paper No. 2 of Wattman, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components to obtain an extract of Shigesbacquiapubesense ethyl acetate.

[Example 2-5] Preparation of Extra-high Pressure Extract of Shigeys Beckia afubesense

The leaves and stems of the dried Shigesbeckia afubensense were pulverized with a mixer, and 1 g of a sample of Shigesbeckia fubesense ground and 76 ml of 18% ethanol were sealed in a polyethylene pack and sealed. Then, (Frescal MFP-7000; Mitsubishi Heavy Industries, Tokyo, Japan). Extraction pressure was 320 MPa and extraction time was 5 min. The extracted sample was filtered with a filter paper No. 2 of Wattman, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components to obtain an extract of Shigesbeckia fubesense ultrahigh pressure.

[ Example  3] Shigesbekkia Oriental lease  Preparation of extract

[Example 3-1] Preparation of ethanol extract of Shigeysubekia orientalis

The leaves and stems of the dried Shigesbeckia orientalis were pulverized with a mixer, and then 100 g of the pulverized Shigesbeckia orientalis sample was added to 1 L of ethanol and extracted at room temperature for 48 hours. The extracted sample was filtered with filter paper of Whatman No. 2, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components to obtain a Shigesbeckia orientalis ethanol extract.

[Example 3-2] Preparation of methanol extract of Shigeys Beckia orientalis

The leaves and stem of the dried Shigesbeckia orientalis were pulverized with a mixer, and 100 g of the pulverized Shigesbeckia orientalis sample was added to 1 L of methanol and extracted with stirring at 50 DEG C for 60 minutes. The extracted sample was filtered with a filter paper No. 2 of Wattman, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components to obtain a methanol extract of Shigesbeckia orientalis.

[Example 3-3] Preparation of hot water extract of Shigesbeckia orientalis

The leaves and stems of the dried Shigesbeckia orientalis were pulverized with a mixer, and then 100 g of the pulverized Shigesbeckia orientalis sample was added to 1 L of water and extracted with stirring at 100 DEG C for 4 hours. The extracted sample was filtered with a filter paper No. 2 of Wattman, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components to obtain a hydrogel extract of Shigesbeckia orientalis.

[Example 3-4] Preparation of an extract of Shigeysubekia orientalis ethyl acetate

The leaves and stems of the dried Shigesbeckia orientalis were pulverized with a mixer, and 100 g of the pulverized Shigusbeckia orientalis sample was added to 1 L of ethyl acetate and extracted with stirring at 50 DEG C for 60 minutes. The extracted sample was filtered with filter paper No. 2 of Wattman, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components to obtain a Shigesbeckia orientalis ethyl acetate extract.

[Example 3-5] Preparation of Extra-high Pressure Extract of Shigeys Beckia Orientalis

The leaves and stems of the dried Shigesbeckia orientalis were pulverized with a mixer and then 1 g of the shiga beccia orientalis sample and 76 ml of 18% ethanol were sealed in a polyethylene pack and sealed. Then, (Frescal MFP-7000; Mitsubishi Heavy Industries, Tokyo, Japan). Extraction pressure was 320 MPa and extraction time was 5 min. The extracted sample was filtered with filter paper No. 2 of Wattman, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components to obtain a Shigesbeckia orientalis extract.

[ Example  4] Pyrenyl  Separation and structure determination

[Example 4-1] Separation of the Kirenol

The concentrated Shigesbeckia orientalis ethanol extract obtained in Example 3-1 was placed in a column packed with silica gel and dissolved in a solvent system in which ethyl acetate and methanol were mixed at a ratio of 10: 0.5 (v / v) Lt; / RTI > Each fraction was divided into seven fractions according to the above sorting order, and the fractions were concentrated and dried. Six fractions (fraction 6) of the seven fractions were collected with developing solvent 10% ethyl acetate using RP-18 reverse phase column chromatography (Lichroprep RP-18 25-40 um, Merck & Co., Whitehouse Station, NJ, USA) . The fractions were divided into two fractions according to the above sorting order, and the respective fractions were concentrated and dried. The fraction 2 (fraction 6-2) of the two fractions was concentrated to dryness, and further recovered by developing solvent 20% ethyl acetate using Rp-18 reverse phase column chromatography. The fractions were divided into three fractions according to the above sorting order, and the respective fractions were concentrated and dried. Finally, the fraction 2 of the three fractions (fraction 6-2-2) was concentrated to dryness to isolate the pure single active substance.

[Example 4-2] Determination of the structure of the quinolone

¹ H-NMR spectrum and ¹³ C-NMR spectrum were measured at 500 MHz and 125 MHz (solvent: MeOH), respectively, in order to determine the structure of the single active material isolated in Example 4-1. The ¹ H ¹ H COSY spectrum and ¹ H ¹³ C HSQC spectra in order to obtain the ¹ H-NMR measurement of correlation between the spectrum and the ¹³ C-NMR correlation of the ¹ H ¹ H on the basis of the result of the spectrum related to the ¹ H ¹³ C relationships And the carbon signal was discriminated by the wavelength emitted from the carbon resonance, and the result was measured.

FAB-MS was measured for mass analysis of the separated single substance. This compound was found to have a molecular weight of 338.48 and a molecular formula of C ₂₀ H ₃₄ O ₄ [M] in FAB-MS, observed at m / z 338.48.

(Wang JP et al., Pharmacogn. Mag., 8: 149-155, 2012) were compared with the results of ¹ H-NMR, ¹³ C-NMR and FAB- As a result, a single substance isolated from the above was identified as a kirenol compound represented by the following formula (2).

(2)

[ Example  5] Chirenol  Enhancement of osteoblast differentiation and inhibition of osteoclast formation by treatment

[Example 5-1] Increase in osteoblast differentiation gene expression of choline

MC3T3-E1 osteoblast cells (ATCC, Manassas, VA, USA) were cultured in α-MEM (α-modified Eagle's Medium) medium containing 10% fetal bovine serum, And placed in a 6-well microtiter plate at 1,00,000 cells / well. (Sigma, St. Louis, Mo., USA) and 10 mM β-glycerol phosphate disodium salt hydrate (Sigma) were added to a culture medium containing 50 μg / ml L-ascorbic acid The osteoblast cells were treated with 10, 20, and 40 μM novolac, respectively, to induce osteoblast differentiation for 3 days. The group not treated with chironol served as a control. RT-PCR was performed to investigate the mRNA expression levels of the major genes ALP, ColA1 and OPN involved in osteoblast differentiation. Total RNA was harvested from the differentiated cells using TRIzol reagent (Invitrogen, Carlsbad, Calif., USA) and reverse transcribed. RT-PCR analysis was performed as follows. First, the RNA was reverse transcribed with reverse transcriptase for cDNA synthesis. RT-PCR was performed with the following primers:

beta -actin:

SEQ ID NO: 1: 5'-AGCCATGTACGTAGCCATCC-3 '(Forward primer)

SEQ ID NO: 2: 5'-CTCTCAGCTGTGGTGGTGAA-3 '(Reverse primer)

ALP:

SEQ ID NO: 3: 5'-CCATGGTAGATTACGCTCACA-3 '(Forward primer)

SEQ ID NO: 4: 5'-ATGGAGGATTCCAGATACAGG-3 '(reverse primer)

ColA1:

SEQ ID NO: 5: 5'-ACTCAGCCGTCTGTGCCTCA-3 '(Forward primer)

SEQ ID NO: 6: 5'-GGAGGCCTCGGTGGACATTA-3 '(Reverse primer)

OPN:

SEQ ID NO: 7: 5'-GACCACATGGACGACGATG -3 '(Forward primer)

SEQ ID NO: 8: 5'-TGGAACTTGCTTGACTATCGA-3 '(Reverse primer)

Also, based on the amount of mRNA expression measured by RT-PCR, the relative mRNA expression level by the treatment with respect to the control group was calculated.

As a result, as shown in FIG. 1, it was confirmed that mRNA expression level of ALP, ColA1 and OPN, which are differentiation genes of osteoblast differentiation, was increased (** p <0.01). This implies that in the MC3T3-E1 osteoblast, the quinolone is effective in promoting bone formation.

[Example 5-2] Increase in activity of alkaline phosphatase (ALP), which is an early differentiation marker of osteoblast cell of chironol

MC3T3-E1 osteoblast differentiated in the same manner as in Example 5-1 was washed with PBS (phosphate buffered saline) solution, and then the cell membrane was dissolved using 0.2% Triton X-100. ALP activity was measured using a SensoLyte p-nitrophenyl phosphate (p-NPP) ALP activity assay kit (AnaSpec, Inc., Fremont, CA, USA). Protein content was determined by Bradford method using Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). ALP activity was expressed as ng / mg protein.

As a result, as shown in FIG. 2, cholineol increased ALP activity in a concentration-dependent manner and showed a significant difference (* p <0.05, ** p <0.01) when compared with the control group. This means that the ability to promote osteoblast differentiation is enhanced by increasing ALP activity, which is an early differentiation marker of osteoblast of the present invention.

[Example 5-3] Improvement of osteoblast mineralization of chironol

MC3T3-E1 osteoblast differentiated for 14 days in the same manner as in Example 5-1 was washed with PBS solution and fixed with 70% ethanol for 1 hour at room temperature. After staining the cells, they were stained with Alizarine Red solution (40 mM, pH 4.2) for 10 minutes, washed 5 times with 3 times distilled water and then added with 0.1 N sodium hydroxide (NaOH) to extract Alizarine Red staining. Respectively. The degree of mineralization was expressed as a percentage (%) as compared to the control group not treated with chironol.

As a result, as shown in FIG. 3, it was confirmed that the mineralization of osteoblast was increased by treatment with chelolnol as compared with the control group. This means that the kinerol of the present invention promotes the differentiation of osteoblasts and has the activity of promoting mineralization.

[Example 5-4] Increased expression of OPG (osteoprotegerin) and RANKL (receptor activator of nuclear factor kappa-B ligand) inhibiting osteoclastogenesis of choline

Total RNA was harvested from MC3T3-E1 osteoblast differentiated in the same manner as in Example 5-1 and reverse transcribed, and RT-PCR was performed with the following primers:

RANKL:

SEQ ID NO: 9: 5'-CGCTCTGTTCCTGTACTTTCGAGCG-3 '(Forward primer)

SEQ ID NO: 10: 5'-TCGTGCTCCCTCCTTTCATCAGGTT-3 '(Reverse primer)

OPG:

SEQ ID NO: 11: 5'-AAAGCACCCTGTATAAAACA-3 '(Forward primer)

SEQ ID NO: 12: 5'-CCGTTTTATCCTCTCTACACTC-3 '(reverse primer)

Also, based on the amount of mRNA expression measured by RT-PCR, the relative mRNA expression level by the treatment with respect to the control group was calculated.

As shown in FIG. 4, OPEN mRNA expression, which inhibits osteoclastogenesis, was significantly increased and rANKL mRNA expression was statistically significantly decreased (P <0.01 ). Therefore, it implies that osteoclast formation is suppressed by controlling the expression of OPG and RANKL, which inhibit osteoclastogenesis, in MC3T3-E1 osteoblasts.

[Example 5-5] Promotion of activity of bone morphogenic protein (BMP) signal transduction system of choline

Total RNA was harvested from MC3T3-E1 osteoblast differentiated in the same manner as in Example 5-1 and reverse transcribed, and RT-PCR was performed with the following primers:

BMP:

SEQ ID NO: 13: 5'-CCAAGACACAGTTCCCTACA-3 '(Forward primer)

SEQ ID NO: 14: 5'-CACGGCTTCTAGTTGATGGA-3 '(Reverse primer)

Runx2:

SEQ ID NO: 15: 5'-GCCGGGAATGATGAGAACTA-3 '(Forward primer)

SEQ ID NO: 16: 5'-TGGGGAGGATTTGTGAAGAC-3 '(Reverse primer)

Osx:

SEQ ID NO: 17: 5'-CTTCCCAATCCTATTTGCCGTTT-3 '(Forward primer)

SEQ ID NO: 18: 5'-CGGCCAGGTTACTAACACCAATCT-3 '(Reverse primer)

Also, based on the amount of mRNA expression measured by RT-PCR, the relative mRNA expression level by the treatment with respect to the control group was calculated.

As shown in FIG. 5, it was confirmed that mRNA expression of BMP2, Runx-related transcription factor 2, and Osx (osterix), which are major genes of osteogenic protein signal transduction system, ** p < 0.01). Therefore, it was confirmed that the activation of osteogenic protein signal transduction system in MC3T3-E1 osteoblasts was promoted by the treatment with pyrenol.

[Example 5-6] Promotion of activation of the Wnt /? -Catenin signal transduction system of choline

Total RNA was harvested from MC3T3-E1 osteoblast differentiated in the same manner as in Example 5-1 and reverse transcribed, and RT-PCR was performed with the following primers:

beta -actin:

SEQ ID NO: 1: 5'-AGCCATGTACGTAGCCATCC-3 '(Forward primer)

SEQ ID NO: 2: 5'-CTCTCAGCTGTGGTGGTGAA-3 '(Reverse primer)

LRP5:

SEQ ID NO: 19: 5 '-AAGGGTGCTGTGTACTGGAC-3' (Forward primer)

SEQ ID NO: 20: 5 '-AGAAGAGAACCTTACGGGACG-3' (Reverse primer)

DVL2:

SEQ ID NO: 21: 5'-GCTTCCACATGGCCATGGGC-3 '(Forward primer)

SEQ ID NO: 22: 5'-TGGCACTGCTGGTGAGAGTCACAG-3 '(Reverse primer)

beta -catenin:

SEQ ID NO: 23: 5'-GCCAAGTGGGTGGTATAGAG-3 '(Forward primer)

SEQ ID NO: 24: 5'-CTGGGTATCCTGATGTGC-3 '(reverse primer)

CCND1:

SEQ ID NO: 25: 5'-AAAATCGTGGCCACCTGGAT-3 '(Forward primer)

SEQ ID NO: 26: 5'-CATCCGCCTCTGGCATTTTG-3 '(Reverse primer)

Also, based on the amount of mRNA expression measured by RT-PCR, the relative mRNA expression level by the treatment with respect to the control group was calculated.

As shown in FIG. 6, it was confirmed that the expression of mRNA of LRP5, DVL2, β-catenin and CCND1, which are major genes of the Wnt / β-catenin signal transduction system, , ** p < 0.01). Therefore, it was confirmed that the activation of Wnt / β-catenin signal transduction system in MC3T3-E1 osteoblasts was promoted by the treatment with the kirenol.

[ Example  6] Recruitment  Enhancement of osteoblast differentiation and inhibition of osteoclast formation by extract treatment

[Example 6-1] Enhancement of Osteoblast Differentiation Gene Expression of Ethanol Extract of Shigae Beckaki Agla Breense, Ethanol Extract of Shigesbeckia afubesense, and Ethanol Extract of Shigae Beckkia Orientalis

The Shigesbeckia agrablasense ethanol extract, Shigeysbeckia afubensense ethanol extract, and Seegesbeckia orientalis ethanol extract prepared in Examples 1-1, 2-1, and 3-1 were each dissolved in a concentration of 10 ppm To examine the mRNA expression levels of the major genes ALP, ColA1, OCN and OPN involved in osteoblast differentiation in MC3T3-E1 osteoblast, the following primers were added after differentiation in the same manner as in Example 5-1 RT-PCR was performed:

OCN:

SEQ ID NO: 27: 5'-CTGAGTCTGACAAAGCCTTC-3 '(Forward primer)

SEQ ID NO: 28: 5'-GCTGCTGTGACATCCATACTTGC-3 '(Reverse primer)

As a result, as shown in Fig. 7, major genes ALP and ColA1 involved in the osteoblast differentiation by the treatment of Shigesbeckia aglablasense ethanol extract, Shigesbeckia afubensense ethanol extract and Shigesbeckia orientalis ethanol extract, , OCN and OPN mRNA expression was increased. Therefore, it was confirmed that the treatment of Shigesbeckia agrablasense ethanol extract, Shigesbeckia afubensense ethanol extract, and Seegesbeckia orientalis ethanol extract promoted differentiation in MC3T3-E1 osteoblasts, thereby promoting bone formation.

[Example 6-2] Expression of OPG that inhibits osteoclast formation of Shigesbeckia aglabresens ethanol extract, Shigesbeckia afubensense ethanol extract, and Shigesbeckia orientalis ethanol extract

The Shigesbeckia agrablasense ethanol extract, Shigeysbeckia afubensense ethanol extract, and Seegesbeckia orientalis ethanol extract prepared in Examples 1-1, 2-1, and 3-1 were each dissolved in a concentration of 10 ppm RT-PCR was performed after differentiation in the same manner as in Example 5-4 to examine the amount of OPG mRNA expression inhibiting osteoclast formation in MC3T3-E1 osteoblast.

As a result, as shown in Fig. 8, the OPG mRNA which inhibits osteoclast formation in osteoblasts by the treatment of Shigesbeckia aglablasense ethanol extract, Shigesbeckia afubensense ethanol extract and Shigesbeckia orientalis ethanol extract It was confirmed that the expression was increased. Thus, the expression of OPG, which inhibits osteoclast formation in MC3T3-E1 osteoblasts, is regulated by the treatment of Shigesbeckia aglabrense ethanol extract, Shigesbeckia afubensense ethanol extract, and Seegesbeckia orientalis ethanol extract. It has a function of inhibiting the osteoclast formation.

[Example 6-3] Effect of Methanol Extract, Hot Water Extract, and Ethyl Acetate Extract of Shigesbeckia Orientalis on Osteoblast Differentiation

The methanol extract, hot-water extract and ethyl acetate extract of Shigesbeckia orientalis prepared in Examples 3-2 to 3-4 were treated at a concentration of 10 ppm, respectively, so that MC3T3-E1 osteoblast cells were cultured in osteoblast differentiation RT-PCR was performed after differentiation in the same manner as in Example 5-1 to examine mRNA expression levels of major genes ALP and ColA1.

As a result, as shown in FIG. 9, it was confirmed that mRNA expression of major genes ALP and ColA1 involved in osteoclast differentiation was increased by treatment with methanol extract, hot water extract and ethyl acetate extract of Shigesbeckia orientalis. Therefore, it was confirmed that the methanol extract, hydrothermal extract, and ethyl acetate extract treatment of Shigesbeckia orientalis promoted differentiation in MC3T3-E1 osteoblasts to promote bone formation.

[ Example  7] Shigesbekkia Oriental lease  Enhancement of osteoblast differentiation and inhibition of osteoclast formation by ultra high pressure extract treatment

[Example 7-1] Increase in osteoblast differentiation of Shigusubekia orientalis ultra-high pressure extract

The Siegesvecia orientalis extract prepared in Example 3-5 was treated at a concentration of 10, 20, and 40 ppm, and the resultant was differentiated in the same manner as in Example 5-1. RT-PCR was performed. Also, based on the amount of mRNA expression measured by RT-PCR, the relative mRNA expression level by the treatment with respect to the control group was calculated.

As a result, as shown in FIG. 10, it was confirmed that mRNA expression of the major genes ALP, ColA1, OCN and OPN involved in the osteoblast differentiation was statistically significantly increased by treatment with Seqsbeckia orientalis extract ( * p < 0.05, ** p < 0.01). This means that the treatment of Shigesbeckia orientalis ultra-high pressure extract promotes bone formation by promoting differentiation in MC3T3-E1 osteoblasts.

[Example 7-2] Increasing activity of alkaline phosphatase (ALP) as an early differentiation marker of osteoblast cell of ultra-high pressure extract of Shigesbeckia orientalis

The Siegesvecia orientalis extract prepared in Example 3-5 was treated at a concentration of 10, 20, and 40 ppm, and differentiated in the same manner as in Example 5-1. Thereafter, the same method as in Example 5-2 To measure ALP activity. As a result, as shown in FIG. 11, the extract of Shigusbeckia orientalis showed a significant concentration-dependent increase in ALP activity and a significant difference when compared with the control (** p <0.01). This means that the Shigesbeckia orientalis extract of the present invention has an excellent ability to promote osteoblast differentiation by increasing ALP activity as an early osteoblast differentiation marker.

[Example 7-3] Improvement of osteoblast mineralization of Shigesbeckia orientalis extract

The Seq. Beckia orientalis extract prepared in Example 3-5 was treated at a concentration of 10, 20, and 40 ppm, and then subjected to differentiation in the same manner as in Example 5-1, followed by the same method as in Example 5-3 To measure the mineralization. As a result, as shown in Fig. 12, it was confirmed that the osteoblast mineralization was significantly increased by treatment with Seqsbeckia orientalis extract (** p <0.01) as compared with the control group. This means that the Shigesbeckia orientalis extract of the present invention has an activity of promoting the differentiation of osteoblasts to promote mineralization.

[Example 7-4] Expression effect of OPG and RANKL inhibiting osteoclast formation of Shigusubekia orientalis ultra-high pressure extract

The Siegesvecia orientalis extract prepared in Example 3-5 was treated at a concentration of 10, 20 and 40 ppm, and after differentiation in the same manner as in Example 5-1, the same method as in Example 5-4 RT-PCR was performed. Also, based on the amount of mRNA expression measured by RT-PCR, the relative mRNA expression level by the treatment with respect to the control group was calculated.

As a result, as shown in FIG. 13, OPG mRNA expression inhibiting osteoclast formation was statistically significantly increased and RANKL mRNA expression was statistically decreased by treatment with Seqsbeckia orientalis ultra-high pressure extract (** p < 0.01). Therefore, it implies that osteoclast formation is suppressed by controlling the expression of OPG and RANKL, which inhibit osteoclastogenesis, in MC3T3-E1 osteoblasts due to treatment with Seqsbeckia orientalis ultra-high pressure extract.

[Example 7-5] Promotion of osseointegration protein signal transduction activity of Shigesbeckia orientalis extract

The Siegesvecia orientalis extract prepared in Example 3-5 was treated at a concentration of 10, 20 and 40 ppm, and after differentiation in the same manner as in Example 5-1, the same method as in Example 5-5 RT-PCR was performed. Also, based on the amount of mRNA expression measured by RT-PCR, the relative mRNA expression level by the treatment with respect to the control group was calculated.

As a result, as shown in Fig. 14, it was confirmed that mRNA expression of BMP2, Runx2, and Osx, which are major genes of osteogenic protein signal transduction system, was significantly increased by treatment with Seqsbeckia orientalis extract (** p &Lt; 0.01). Therefore, it was confirmed that osteogenic protein signal transduction activity is promoted in MC3T3-E1 osteoblast by treatment with Seqsbeckia orientalis ultra-high pressure extract.

[Example 7-6] Promotion of activity of Wnt /? -Catenin signal transduction system of Shigesbeckia orientalis ultra-high pressure extract

The Seq. Beckia orientalis extract prepared in Example 3-5 was treated at a concentration of 10, 20, and 40 ppm, and after differentiating in the same manner as in Example 5-1, the same method as in Example 5-6 RT-PCR was performed. Also, based on the amount of mRNA expression measured by RT-PCR, the relative mRNA expression level by the treatment with respect to the control group was calculated.

As a result, as shown in FIG. 15, mRNA expression of LRP5, DVL2, β-catenin and CCND1, which are major genes of the Wnt / β-catenin signal transduction system, was statistically increased by treatment with Seqsbeckia orientalis extract (* P < 0.05, ** p < 0.01). Therefore, it was confirmed that the treatment of Shigesbeckia orientalis ultra-high pressure extract promotes the activity of Wnt / β-catenin signaling system in MC3T3-E1 osteoblasts.

Hereinafter, an example of preparation of foods and medicines for preventing, treating or improving osteoporosis, which comprises as an active ingredient the choline or extract of Shigesbeckia orientalis containing the same according to the present invention will be explained. But rather just to explain it specifically. The present invention relates to a method for the prevention and / or treatment of osteoporosis, which comprises administering to a mammal a therapeutically effective amount of a compound of formula The compositions were prepared according to conventional methods.

[Manufacturing Example 1] Production of food

[Manufacturing Example 1-1] Preparation of health food

Vitamin E acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 ug of vitamin B12, 10 mg of vitamin C , 10 ug of biotin, 1.7 mg of nicotinic acid amide, 50 ug of folate, 0.5 mg of calcium pantothenate, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of monobasic potassium phosphate, The above ingredients may be mixed according to a conventional method for preparing healthy foods, and then granules of granules may be prepared by mixing granules Can be prepared and used in the manufacture of a health food composition according to a conventional method.

[Production Example 1-2] Preparation of Healthy Drink

Purified water was added to each of the extracts of Examples 1 to 3 or 1000 mg of quinolin, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of a plum concentrate and 1 g of taurine, After mixing, the mixture is stirred and heated at 85 for about 1 hour, and then the resulting solution is filtered and sterilized in a sterilized 2 L container, which can then be stored in a refrigerator for use in the manufacture of a health beverage composition.

[Production Example 1-3] Chewing gum

Chewing gum was prepared in a conventional manner by mixing 20% by weight of a gum base, 76.9% by weight of sugar, 1% by weight of fragrance and 2% by weight of water and a scavenger extract of the above Examples 1 to 3 or 0.1%

[Manufacturing Example 1-4] Candy

Candy was prepared by mixing 60% by weight of sugar, 39.8% by weight of starch syrup, 0.1% by weight of fragrance, and the extract of Heliothiazide of Examples 1 to 3 or 0.1% by weight of Kirenol.

[Manufacturing Example 1-5] Biscuits

A mixture of 0.75% by weight of sodium chloride, 0.78% by weight of glucose, 11.78% by weight of palm shortening, 1.54% by weight of ammonium, 0.17% by weight of sodium bicarbonate and 0.16% by weight of sodium bisulfite, 25.59% by weight of Grade I, 22.22% 1.45 wt% of rice flour, 0.0001 wt% of vitamin B, 0.04 wt% of milk fractions, 20.6998 wt% of water, 1.16 wt% of whole milk powder, 0.29 wt% of replacement milk powder, 0.03 wt% of calcium phosphate, 0.29 wt% 7.27% by weight of the nutrients and the excipient extract or wt% of the quinolone of Examples 1 to 3 were mixed to prepare biscuits by a conventional method.

[Manufacturing Example 2] Production of medicines

[Manufacturing Example 2-1]

The excipient extracts of Examples 1 to 3, or 50 mg of calenol and 2 g of crystalline cellulose were mixed and filled in an airtight container according to a conventional acid preparation method to prepare powders.

[Manufacturing Example 2-2] Purification

50 mg of quinolol, 400 mg of crystalline cellulose, and 5 mg of magnesium stearate were mixed with the extracts of Examples 1 to 3, or tablets were prepared by tableting according to a conventional preparation method.

[Production Example 2-3]

The excipient extracts of Examples 1 to 3 or 30 mg of quinolone, 100 mg of whey protein, 400 mg of crystalline cellulose, and 6 mg of magnesium stearate were mixed and filled in gelatin capsules according to a conventional capsule preparation method to prepare capsules Respectively.

[Production Example 2-4] Injection

The active ingredient was dissolved in distilled water for injection and the pH was adjusted to about 7.5. Then, the extracts of Examples 1 to 3, 100 mg of quinolone, distilled water for injection, and pH adjusting agent were mixed to prepare 2 ml Dose ampoule and sterilized to prepare an injection.

<110> University-Industry Foundation, Yonsei University <120> Composition for prevention, improvement or treatment of          osteoporosis comprising kirenol or extract of Sigesbeckia spp. <130> P14U16C0906 <150> 2013-0084905 <151> 2013-07-18 <160> 28 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin forward primer <400> 1 agccatgtac gtagccatcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin reverse primer <400> 2 ctctcagctg tggtggtgaa 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ALP forward primer <400> 3 ccatggtaga ttacgctcac a 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ALP reverse primer <400> 4 atggaggatt ccagatacag g 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ColA1 forward primer <400> 5 actcagccgt ctgtgcctca 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ColA1 reverse primer <400> 6 ggaggcctcg gtggacatta 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> OPN forward primer <400> 7 gaccacatgg acgacgatg 19 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> OPN reverse primer <400> 8 tggaacttgc ttgactatcg a 21 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RANKL forward primer <400> 9 cgctctgttc ctgtactttc gagcg 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RANKL reverse primer <400> 10 tcgtgctccc tcctttcatc aggtt 25 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OPG forward primer <400> 11 aaagcaccct gtataaaaca 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OPG reverse primer <400> 12 ccgttttatc ctctctacac tc 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BMP forward primer <400> 13 ccaagacaca gttccctaca 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BMP reverse primer <400> 14 cacggcttct agttgatgga 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Runx2 forward primer <400> 15 gccgggaatg atgagaacta 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Runx2 reverse primer <400> 16 tggggaggat ttgtgaagac 20 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Osx forward primer <400> 17 cttcccaatc ctatttgccg ttt 23 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Osx reverse primer <400> 18 cggccaggtt actaacacca atct 24 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRP5 forward primer <400> 19 aagggtgctg tgtactggac 20 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LRP5 reverse primer <400> 20 agaagagaac cttacgggac g 21 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DVL2 forward primer <400> 21 gcttccacat ggccatgggc 20 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DVL2 reverse primer <400> 22 tggcactgct ggtgagagtc acag 24 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-catenin forward primer <400> 23 gccaagtggg tggtatagag 20 <210> 24 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> beta-catenin reverse primer <400> 24 ctgggtatcc tgatgtgc 18 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCND1 forward primer <400> 25 aaaatcgtgg ccacctggat 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCND1 reverse primer <400> 26 catccgcctc tggcattttg 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OCN forward primer <400> 27 ctgagtctga caaagccttc 20 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OCN reverse primer <400> 28 gctgctgtga catccatact tgc 23

Claims (18)

A pharmaceutical composition for preventing or treating osteoporosis, comprising a compound represented by the following formula (1) as an active ingredient:
[Chemical Formula 1]

The pharmaceutical composition for preventing or treating osteoporosis according to claim 1, wherein the compound represented by the formula (1)
(2)

[Claim 2] The compound according to claim 1, wherein the compound represented by the formula (1) is one or more extracts or fractions thereof selected from the group consisting of Shigesbeckia glabresense, Shigesbeckia afubesense and Shigesbeckia orientalis For the prevention or treatment of osteoporosis. A food composition for preventing or ameliorating osteoporosis comprising a compound represented by the following formula (1) as an active ingredient:
[Chemical Formula 1]

The composition for preventing or improving osteoporosis according to claim 4, wherein the compound represented by the formula (1) is represented by the following formula (2).
(2)

[Claim 4] The compound according to claim 4, wherein the compound represented by the formula (1) is one or more extracts or fractions thereof selected from the group consisting of Shigesbeckia glabresense, Shigesbeckia afubesense and Shigesbeckia orientalis Or a pharmaceutically acceptable salt thereof.


delete delete delete delete delete delete delete delete delete delete delete delete

Images