KR101885667B1 - Extract of Oryza sativa for promoting osteoblast differentiation and its use - Google Patents

Abstract

The rice paddy extract of the present invention increases the expression of ALP, Osterix and Runx2, which are genes involved in the differentiation of osteoblasts, (BMD) in an ovariectomized osteoporosis animal model, decreases fat cells in the bone marrow, decreases the expression of PPARγ, aP2, and CD36, which are genes involved in cell differentiation, It can be used as an active ingredient of functional foods.

Description

Extract of Oryza sativa for promoting osteoblast differentiation and inhibiting adipocyte differentiation and its use

The present invention relates to a rice extract which promotes osteoblast differentiation and inhibits adipocyte differentiation. The rice extract of the present invention can be used for the production of health functional foods or medicines related to bone metabolism diseases or obesity.

The bone not only supports the muscles and organs structurally, but also supports the body's soft tissues and body weight, surrounds the internal organs, protects the internal organs from external impacts, stores calcium or other essential minerals such as phosphorus or magnesium It is one of the important parts of the human body.

Bones are composed of osteoblasts, osteocytes, and osteoclasts. Among them, osteoblast is derived from mesenchymal stem cell which can differentiate into cartilage (contract), myocyte, and adipocyte, and forms bone tissue through a proliferating, bone matrix forming, . The osteoclasts also play a role in absorbing bone.

Adult bone is removed by old osteoclasts, and the bone formation process takes place by continuously repeating bone resorption and production, replacing old bone with new bone by osteoblasts. For example, osteoblast cells regulate the differentiation of osteoclasts responsible for bone resorption through secretion of substances such as RANKL (receptor activator of nuclear factor-κB ligand) and its inducible receptor, OPG (osteoprotegerin) Lt; / RTI >

When the homeostasis of the bone metabolism is destroyed by a specific cause, bone metabolic diseases such as osteoporosis, osteoporosis, or fracture occur.

Osteoporosis, a typical bone metabolic disorder, is characterized by a decrease in bone mass and bone quality, a bone mineral density of 2.5 or less, or a T-score of less than -2.5 (average deviation from normal bone mass in normal adults) State. Osteoporosis is a frequent occurrence in postmenopausal women and as the age increases, the bone matrix decreases and the fat cells form in the void space. The formed fat cells decrease the functions and differentiation of bone-forming osteoblasts and the inflammatory cytokines And is known to promote the function and differentiation of osteoclasts that are responsible for bone resorption. Excessive reduction in BMD can easily lead to fractures in small impacts. Osteoporosis can not lead to a healthy life by restricting long-term activities due to various fractures caused by weakening of the bones, especially femur fracture or vertebral fracture, rather than the symptom itself, resulting in 15% of the elderly death It is known.

Osteodystrophy is also called bone dystrophy and is a bone disease caused by chronic renal failure. It is born by abnormally abnormal kidney function, and when the kidney is weak, it does not dialyse. This kind of bone disease is called renal osteodystrophy. Bone diseases associated with osteophagia include osteomalacia fibrous osteitis (osteitis fibrosa).

For the treatment or prevention of bone metabolic diseases, calcium supplement is recommended, and for menopausal women, vitamin D, or hormones such as estrogen or calcitonin are recommended. In addition, bone resorption inhibitors such as bisphosphonates such as Fosamax (alendronate) and Actonel (ingredient name: risedronate), which inhibit osteoclasts and induce death, are mainly used.

However, calcium supplementation inhibits the secretion of parathyroid hormone and prevents bone loss due to bone resorption. However, it is known that individual differences in bone mass maintenance are severe. Hormones increase bone mineral density, but side effects such as breast cancer, myocardial infarction and venous thrombosis (Nelson, HD et al., JAMA, 288: 872-881, 2002; Lemay, A., J.Obstet. Bynaecol. Can., 24: 711-7152-3). In the case of bisphosphonate preparations, cases of jawbone necrosis, severe atrial fibrillation, inability to bones or joints, or pain in the musculoskeletal system have been reported (Coleman RE., Br J Cancer, 98: 1736-1740 (2008).

In addition, the agents for treating or preventing the bone diseases described above have pharmacological actions to prevent bone loss, but they have not had the effect of restoring the reduced bone mass to the original state.

Accordingly, the inventors of the present invention have confirmed that rice extract can treat and prevent bone metabolic diseases or obesity through the regulation of differentiation of osteoblasts and adipocytes, and completed the invention.

It is an object of the present invention to provide a health functional food for improving or preventing bone metabolic diseases comprising rice extract as an active ingredient.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of bone metabolic diseases comprising rice extract as an active ingredient.

The health functional food for improving or preventing bone metabolic diseases of the present invention contains rice ( Oryza sativa ) extract as an active ingredient.

In addition, the pharmaceutical composition for preventing or treating bone metabolic diseases of the present invention contains rice ( Oryza sativa ) extract as an active ingredient.

The term " rice " used in the present invention includes the seeds of the step of growing the seedlings in which roots are produced from rice seed. The rice paddy extract is an outpost, stem or root extract of rice, and is preferably a root extract. The outposts refer to whole plants, including roots and stems, except for grain. The root refers to a portion of rice except for the ground portion.

The rice root extract of the present invention has the highest osteoblast differentiation activity and adipocyte differentiation inhibitory activity in the root extract of the harvester. The term " harvesting time " used in the present invention means 50 days before harvesting of rice, preferably 30 days before harvest to a certain period after harvest. The time after harvest is not particularly limited, but it can be used within one year after harvest.

In addition, the osteoblast differentiation activity and the adipocyte differentiation inhibiting activity of the rice root extract tended to increase with an increase in the growth period. However, rice roots remaining in the rice paddy after commercial harvesting Alternatively, it may be somewhat less active to utilize roots that can be obtained within weeks or within 1 to 2 months after growth in the matrix, but this is advantageous in reducing production costs.

The extract may be water, an organic solvent, or a mixed solvent thereof, including purified water. Preferably, the organic solvent may be at least one solvent selected from the group consisting of alcohols having 1 to 4 carbon atoms, hexane, dichloromethane, ethyl acetate, acetone, chloroform and diethyl ether, more preferably methanol, ethanol or methanol And an aqueous solution of ethanol. In addition to the organic solvent, polar solvents such as acetic acid, dimethyl-formamide (DMFO) and dimethyl sulfoxide (DMSO), acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, 2,2,4-trimethylpentane, decane, Butene, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, Non-polar solvents such as benzene, diethyl ether, diethyl sulfide, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride and THF (Tetrahydrofuran) may be used.

The term 'extract' used in the present invention includes fractions obtained by further fractionating the above extract. That is, not only the water, the lower alcohol having 1 to 4 carbon atoms, the nonpolar solvent, or the mixed solvent thereof, but also the fractions fractionated by the above-mentioned solvents, or the ultrafiltration membrane having various molecular weight cutoff values And fractions obtained.

Preferably, the rice extract of the present invention is an extract of water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, or a fraction thereof obtained by fractionation with dichloromethane or ethyl acetate.

The rice extract of the present invention contains at least one compound selected from the following chemical formulas (1), (2) and (3) as an active ingredient or indicator component which promotes osteoblast differentiation and inhibits adipocyte differentiation.

The method for producing the extract of the present invention can be carried out by, for example, shredding the rice seeds, stem or root, shrunk, and then adding about 1 to 50 times, preferably about 10 to 40 times the weight of the dried sample, Organic solvent or a mixed solvent thereof at a temperature of 20 to 100 ° C., preferably 40 to 60 ° C. for about 24 hours or for 2 to 4 hours under stirring, hot-water extraction, cold extraction, Or an extract method such as supercritical extraction, preferably extracting hot water, and extracting the obtained extract, by filtration, concentration under reduced pressure or drying to obtain the extract of the present invention.

The term " as an active ingredient " used in the present invention means an amount sufficient to achieve the efficacy or activity of the rice extract.

The bone metabolic diseases include osteoporosis, osteoporosis, or fracture.

The 'osteoporosis' includes clinical classification according to the measurement of bone mineral density (BMD), that is, osteopenia, osteoporosis, and severe osteoporosis. It also includes primary osteoporosis (type 1 postmenopausal osteoporosis), type 2 senile osteoporosis, and secondary osteoporosis.

The 'osteodystrophy' includes osteomalacia, osteitis fibrosa, and the like.

The term 'fracture' refers to a state in which continuity of bone or cartilage is completely or incompletely lost or a linear deformation occurs. The fracture is defined as an anatomic location, the degree of fracture, the direction of the fracture surface, the presence of open window, the number of fracture fragments, stability, dislocation of the fracture fragment, pathologic fractures caused by osteoporosis and tumor osteomyelitis, And fatigue fractures that are caused by the application of a force.

The health functional food for improving or preventing bone metabolic diseases of the present invention promotes osteoblast differentiation and has an activity of inhibiting adipocyte differentiation. Thus, obesity and bone metabolic diseases can be simultaneously improved or prevented.

The health functional food for improving or preventing bone metabolic diseases according to the present invention may include a rice extract and a vitamin, a natural product, or an extract thereof having an activity of improving or preventing bone metabolic diseases. For example, one or more of the calcium enhancer, vitamin D, and the like may be included in combination with the rice extract.

The health functional food for improving or preventing bone metabolic diseases according to the present invention may further comprise, in addition to the above-described active ingredients, a food-acceptable food-aid additive.

The health functional food of the present invention includes various foods, gums, tea, vitamin complex, health supplement foods and the like, and can be used as powder, granule, tablet, capsule or beverage.

When the rice extract is added to a food or beverage for the purpose of improving or preventing bone metabolic diseases, the amount of the rice extract in the food is generally from 0.001 to 5% by weight of the total food, Can be added at a ratio of 0.002 to 5 g, preferably 0.03 to 1 g, based on 100 ml.

The beverage is not particularly limited to the liquid ingredient other than the rice extract as an active ingredient, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As natural flavorings other than those described above, natural flavoring agents (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) The ratio of natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 mL of beverage.

In addition to the above, the health functional food of the present invention may contain flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional foods of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so important, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the health functional food of the present invention.

The pharmaceutical composition for preventing or treating bone metabolic diseases of the present invention promotes osteoblast differentiation and has an activity of inhibiting adipocyte differentiation. Thus, obesity and bone metabolic diseases can be prevented or treated at the same time.

The pharmaceutical composition for prevention or treatment of bone metabolic diseases according to the present invention may contain drugs, vitamins, natural products, or extracts thereof, which have an activity of preventing or treating bone metabolism diseases, together with the above rice extract. For example, a combination of the rice extract and a bone resorption inhibitor such as a calcium enhancer, vitamin D, a hormone such as estrogen or calcitonin, a bisphosphonate system such as Fosamax (alendronate), Actonel (ingredient name: risedronate) May be included in combination.

The pharmaceutical composition for preventing or treating bone metabolic diseases according to the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredient. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, Other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The pharmaceutical composition for preventing or treating bone metabolic diseases according to the present invention may be administered orally or parenterally (for example, intravenously, intraperitoneally, subcutaneously or topically) depending on the purpose, Sex, health condition, diet, time of administration, administration method, excretion rate and severity of the disease. In the present invention, the dosage of the rice extract is 0.1 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg. The administration can be administered once a day or several times depending on the purpose.

The rice paddy extract of the present invention increases the expression of ALP, Osterix and Runx2, which are genes involved in the differentiation of osteoblasts, (BMD) in an ovariectomized osteoporosis animal model, decreases fat cells in the bone marrow, decreases the expression of PPARγ, aP2, and CD36, which are genes involved in cell differentiation, It can be used as an active ingredient of functional foods.

FIG. 1 is a schematic view showing a process for preparing a solvent fraction of a rice extract in Production Example 3. FIG.
FIG. 2 is a graph showing the change of ALP production by -b value after ALP staining to determine the change of ALP (alkaline phosphatase) production by the treatment of rice bark extracts in C3H10T1 / 2 cell line in Experimental Example 1 to be.
FIG. 3 is a graph showing the results of oil staining after oil-red staining in order to show changes in lipid production according to the treatment of each part of rice seedlings during differentiation of adipocytes of C3H10T1 / 2 cell line in Experimental Example 1, .
FIG. 4 is a graph showing changes in ALP production after the ALP (alkaline phosphatase) production by the root extract treatment of rice hulls in the C3H10T1 / 2 cell line in Experimental Example 2. FIG. It is a graph.
FIG. 5 is a graph showing the results of lipid peroxidation of the C3H10T1 / 2 cell line in Experimental Example 2, showing the change in fat production according to the rooting treatment of rice seedlings at different growth stages, Graph.
FIG. 6 is a graph showing the change in ALP production after ALP (alkaline phosphatase) production in each of the solvent fractions treated with the rice root extract in the osteoblast differentiation of C3H10T1 / 2 cell line in Experimental Example 3 It is a graph.
FIG. 7 is a graph showing the results of lipid peroxidation of the C3H10T1 / 2 cell line in Experimental Example 3, showing the change in lipid production according to the treatment with each fraction of the solvent of the rice root extract, Graph.
FIG. 8 is a graph showing changes in ALP production after ALP (alkaline phosphatase) production according to the treatment of 6 compounds isolated in Production Example 4 during osteoblast differentiation of C3H10T1 / 2 cell line in Experimental Example 4, As shown in FIG.
FIG. 9 is a graph showing the changes in lipid production according to the treatment of six compounds isolated in Preparation Example 4 during differentiation of C3H10T1 / 2 cell line in Experimental Example 4. The dyeing results are shown in FIG. .
10 is a graph showing changes in the expression levels of ALP, Osterix and Runx2 in the C3H10T1 / 2 cell line according to the treatment with the six compounds isolated in Production Example 4 in Experimental Example 4. FIG.
FIG. 11 is a graph showing changes in expression levels of CD36, aP2 and PPARγ mRNAs in the C3H10T1 / 2 cell line according to the treatment with 6 single peak compounds isolated in Production Example 3 in Experimental Example 4. FIG.
FIG. 12 is a weight comparison graph of ovariectomized rats in which the root extract of rice was orally administered for 12 weeks in Experimental Example 5. FIG.
FIG. 13 is a graph showing bone mineral density (BMD) of ovariectomized rats treated with rice root extract in Experimental Example 5. FIG.
FIG. 14 is a graph showing the amounts of ALP, Osterix and Runx2 expressed in osteoclasts in the femur of ovariectomized rats to which the rice root extract was administered in Experimental Example 5. FIG.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. The following Examples and Experimental Examples are merely illustrative of the present invention, but the present invention is not limited thereto.

Production Example 1: Production of rice (Oryza sativa) extract

Rice ethanol extracts were prepared by dividing the harvested rice stem, root, algae (except chaff), and outpost (excluding stem and root and whole hog).

Each of the above samples was thoroughly dried at room temperature for 2 days and finely pulverized to obtain a powder sample. Then, 200 ml of a 70 (v / v)% aqueous ethanol solution was added per 10 g of the powder sample, The supernatant was filtered with Wattmann No.1 filter paper, and 200 ml of a 70 (v / v)% ethanol aqueous solution was added to the remaining sample in an amount of 10 g of the sample. The supernatant was again extracted for 24 hours, And filtered in the same manner. The filtrate was concentrated under reduced pressure at 45 [deg.] C with a rotary vacuum evaporator to remove the solvent completely to prepare rice seedling ethanol extract, rice stem ethanol extract, rice seed ethanol extract and rice seed ethanol extract.

Production Example 2: Preparation of root extract of rice (Oryza sativa) according to growing time

Roots were cultivated in the same manner as in Preparation Example 1, except that the roots were classified into roots after 90 days of rooting, root after 110 days of rooting and rooting after 130 days of rooting (after harvesting) Rice root extracts were prepared.

Preparation Example 3 Preparation of Solvent Fraction of Rice (Oryza sativa) Root Extract

The rice seed ethanol extract of Preparation Example 1 was fractionated stepwise as shown in Fig. 1 to prepare respective fractions.

The soluble fraction of the rice seed ethanol extract of Preparation Example 1 was fractionated stepwise with hexane, dichloromethane, ethyl acetate, butyl alcohol and water, and the hexane fraction, the dichloromethane fraction (DCM), the ethyl acetate fraction (EtOAc) Butanol fractions (BuOH) and water fractions (DW) were obtained, respectively, and these were concentrated and lyophilized to use.

Experimental Example 1: Osteoblast differentiation activity and adipocyte differentiation inhibitory activity of rice extract

(1) ALP (alkaline phosphatase) promoting activity of rice extract

The C3H10T1 / 2 cell line derived from mouse embryonic fibroblasts is a pluripotent stem cell line that can differentiate into various cell lines including osteoblasts. One of the characteristics of the osteoblast is that it exhibits ALP (Alkaline phosphatase) activity. Therefore, the osteoblast differentiation effect was measured by ALP activity of C3H10T1 / 2 cell lines.

The C3H10T1 / 2 cell line was cultured in DMEM medium supplemented with 10% FBS, 1% penicillin and streptomycin at 37 ° C in a 5% CO 2 environment. The C3H10T1 / 2 cells were cultured in 6-well plates at a concentration of 2.5 x 10 < 4 > / ml with a medium containing 10 mM [beta] -glycerophosphate and 50 [mu] g / ml ascorbic acid for osteoclast differentiation, 0.1, 0.5, 1 and 5 μM, respectively, and the medium was changed every 3 days and differentiated for 9 days.

C3H10T1 / 2 cells were cultured in DMEM medium supplemented with 10% FBS, 1% penicillin and streptomycine at 37 ° C and 5% CO 2. The cells were cultured in a 6-well plate at a concentration of 2.5 × 10 4 cells / ml together with a medium containing 10 mM β-glycerophosphate and 50 μg / ml ascorbic acid for osteoclast differentiation, and the medium was changed every 3 days. (ALP) staining was performed using 5-Bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium (BCIP / NBT) The result of measuring the Lab color space using the image file of the well plate was shown in FIG.

Strong osteoblast differentiation activity was shown in the outpost, stem and root extracts, and the activity was highest in root extracts and low in oat extracts (see FIG. 2).

(2) Inhibitory activity of adipocyte differentiation on the adipocyte differentiation

After the cells were dispensed into 6 well plates in the same manner as in Experimental Example 1, the medium containing 1 μM dexamethasone, 5 μg / mL insulin and 1 μM troglitazone, and 40 μg / ml of the rice extract of Preparation Example 1 And cultured for a total of 9 days. After completion of the differentiation, the medium was removed, and the cells were fixed with 10% neutral buffered formalin, stained with 0.5% oil red oozing solution, and then the Lab color space was measured using an image file of the well plate. .

The inhibitory activity of adipocyte differentiation was similar to that of the seedling extract and the seed extract, and the effect of inhibiting adipocyte lipogenesis was shown in the order of stem extract and root extract (see FIG. 3).

From the results shown in FIGS. 2 and 3, it was confirmed that the rice root extract had the highest activity of inhibiting osteoblast differentiation and differentiation of adipocytes.

Experimental Example 2: Osteoblast differentiation activity and adipocyte differentiation inhibition activity of rice root according to growth period

The osteoblast differentiation activity and the adipocyte differentiation inhibitory activity of the root extract of Preparation Example 2 after 130 days (after harvest) and after rooting after 90 days of rooting, rooting after 110 days of seedling cultivation, The results are shown in FIGS. 4 and 5. FIG.

(1) ALP (alkaline phosphatase) promoting activity of rice roots according to the growth period

According to FIG. 4, the osteoblast differentiation activity tended to increase with the increase of growth period from rice seed, and there was no significant difference in osteoblast differentiation activity after 110 days of seedling cultivation.

(2) Inhibitory activity of adipocyte differentiation on the root of rice

According to FIG. 5, the activity of inhibiting adipocyte differentiation was increased as the cultivation period was increased from rice cucumber to 130 days after seeding (after harvesting). In particular, 110 days after seeding and 130 days after harvesting ), It was confirmed that the activity of inhibiting adipocyte differentiation was greatly increased as the growth period was increased.

From the results of FIGS. 4 and 5, it was found that it would be advantageous to use the remaining rice roots after harvesting in order to commercialize the rice root extract having the highest activity to inhibit osteoblast differentiation and differentiation of adipocytes.

Experimental Example 3: Osteoblast differentiation activity and adipocyte differentiation inhibitory activity of the rice root fraction

The osteoblast differentiation activity and adipocyte differentiation inhibiting activity of the hexane fraction (Hexane), the dichloromethane fraction (DCM), the ethyl acetate fraction (EtOAc), the butanol fraction (BuOH) and the water fraction (DW) 1, and the results are shown in Figs. 6 and 7. Fig. However, the addition concentration of each sample was changed to 20 μg / ml, and ½ million of them were added.

(1) ALP (alkaline phosphatase) production promoting activity of the fraction

According to FIG. 6, the osteogenic differentiation activity was significantly increased only in the dichloromethane fraction (DCM) and the ethyl acetate fraction (EtOAc), and osteoblast differentiation activity was not observed in the remaining fractions.

(2) Inhibitory activity of adipocyte differentiation activity of fraction

According to FIG. 7, it was confirmed that the dichloromethane fraction (DCM) and the ethyl acetate fraction (EtOAc), especially the dichloromethane fraction (DCM)

Therefore, the active ingredient of the rice root extract having the activity of inhibiting osteoblast differentiation and the differentiation of adipocytes was expected to be present in the dichloromethane fraction, and the active compound was separated from the fraction.

Production Example 4: Isolation of Active Compound from Dichloromethane Fraction (DCM) of Rice Root Extract

From the dichloromethane fraction (DCM) of Preparation Example 3, fractions corresponding to extraction solvent mixing ratios were prepared and separated in order to isolate the active compounds having the activity of differentiating osteoblasts and the activity of inhibiting differentiation of adipocytes. 1 liter of an elution solvent was prepared by mixing chloroform and methanol at a mixing ratio of 100: 1, 50: 1, 10: 1, 5: 1, 2: 1 and 1: 1. C18 silica gel column chromatography -18 silica gel column chromatography, each fraction was obtained by using each of the elution solvents twice.

After confirming the physiological activity of the fraction, the active fraction layer was fractionated according to the molecular size and selected. Each fraction was obtained by Sephadex LH-20 column size-exclusion chromatography using Sephadex LH-20 methanol single solvent. After confirming the physiological activity with the obtained fractions, two active fraction layers of the obtained fractions were confirmed by reprocessing the active fraction layer.

Separation conditions were established by using TLC (thin layer chromatography), and the two fractions were separated by prep-HPLC and confirmed by TLC. The structure of the substance identified by a single peak was analyzed by electrospray ionization-mass spectrometry (ESI-MS), 1H-nuclear magnetic resonance (1H-NMR), and carbon-nuclear magnetic resonance spectroscopy (13C-NMR). In the case of the new compound, 2D NMR was measured to obtain IR, CD and alpha values, and the substance was confirmed.

The compounds of Formulas 1 to 6 were confirmed at a total of 6 peaks by the above method.

Peak 1 is a 2E, 2'E) - (2,5-bis (4-hydroxy-3,5-dimethoxyphenyl) tetrahydrofuran- -hydroxyphenyl) acrylate).

[Chemical Formula 1]

Peak 2 is a (2E, 2'E) - (2,5-bis (4-hydroxy-3,5-dimethoxyphenyl) tetrahydrofuran- 4-hydroxyphenyl) acrylate (isomer).

(2)

Peak 3 is a 2 (2E, 2'E) - (2,5-bis (4-hydroxy-3,5-dimethoxyphenyl) tetrahydrofuran- (4-hydroxyphenyl) acrylate (isomer).

(3)

.

.

Peak 4 was 2-O-feruloyl-glyceride of Formula 4 below.

Peak 5 was 2-O-feruloyl-glyceride (isomer) of Formula 5 below.

Peak 6 was Takakin of Chemical Formula 6 below.

Experimental Example 4: Osteoblast differentiation activity and adipocyte differentiation inhibitory activity of a compound derived from rice root extract

The osteoblast differentiation activity and the adipocyte differentiation inhibiting activity of the compounds of Formulas 1 to 6 of Peaks 1 to 6 isolated in Preparation Example 4 were measured in the same manner as in Experimental Example 1. The results are shown in FIGS. 8 and 9 . However, the addition concentration of each sample was changed to 5 μg / ml, and only 1/8 was added.

(1) ALP (alkaline phosphatase) promoting activity of the compound derived from the root extract of rice

According to FIG. 8, the osteoblast differentiation activity was significantly increased in the compounds of Formulas 1 to 3, particularly the compounds of Formula 1 of Peak 1, and the compounds of Formulas 4 to 6 of the remaining peaks 4 to 6 showed osteoblast differentiation Activity was weak.

(2) Inhibitory activity of adipocyte root-derived compound on adipocyte differentiation

According to FIG. 9, the adipocyte differentiation inhibiting activity of the compounds of Formulas 1 and 2 of peaks 1 and 2 was markedly increased, the adipocyte differentiation inhibiting activity of the compound of Formula 3 of peak 3 was shown to be significantly increased, The compounds of formulas (4) to (6) showed little inhibitory activity on adipocyte differentiation.

Therefore, the active ingredients of the rice root extract having the activity of differentiating osteoblasts and inhibiting the differentiation of adipocytes were predicted as the compounds of the formulas (1) to (3).

Experimental Example 5: Expression of osteoblast differentiation factor and adipocyte differentiation factor of a compound derived from rice root extract

MRNA expression levels of osteoblast differentiation factors and adipocyte differentiation factors in the cells were examined when the compounds of Formulas 1 to 6 of Peaks 1 to 6 isolated in Production Example 4 were treated and are shown in FIGS. 10 and 11, respectively.

First, as in Experimental Example 1, C3H10T1 / 2 cell line was differentiated into osteoblasts and the expression levels of ALP, Ostreix and Runx2, which are osteoblast differentiation factors, were confirmed. In the same manner as in Experimental Example 1, C3H10T1 / 2 cell line was differentiated into adipocytes, and the amounts of adipocyte differentiation factors CD36, aP2 and PPARγ were determined.

Total RNA extraction was performed with TRIzol (Invitrogen). 1 μg of the separated RNA was synthesized by adding random primer, dNTP, and PrimeScript ™ Reverse Transcriptase (TaKaRa). The synthesized cDNA was subjected to realtime PCR with SYBR Premix Ex Taq (TaKaRa) using primers.

The compounds of Formulas 1 to 3 of peaks 1 to 3 all increased the expression levels of ALP, Osterix and Runx2, which are osteogenic differentiation factors, and markedly increased the expression level particularly in the compounds of Formulas 1 and 2 (see FIG. 10) .

Also, the compounds of formulas (1) to (3) of peaks 1 to 3 markedly decreased the expression amounts of CD36 and aP2 and PPARγ, which are adipocyte differentiation-related genes, particularly the expression levels of the compounds of formulas (1) and (2) Reference).

Experimental Example 6: ovariectomy Animal test using an animal model of bone metabolism disease

To investigate the effect of rice extract on the treatment and prevention of bone metabolic diseases, the extracts of rice root extract of Preparation Example 1 were administered to ovariectomized rats and bone mineral density was measured and histological analysis was carried out.

(1) Animal breeding and ovariectomy

8-week-old female SD (Sprague-Dawley) rats were purchased and had a purifying period of one week. At the age of 9 weeks, ovariectomies were performed and he had a one week recovery period. For 12 weeks, the samples were orally administered once a day. The experimental animals were housed in a single cage. The environmental conditions were adjusted to room temperature of 25 ± 5 ° C, relative humidity of 50 ± 10% and illumination time of 12 hours . Feed (AIN-93g) and water were allowed to be ingested freely.

The experimental groups were divided into two groups: sham without ovariectomy, ovariectomized group (OVX) after ovariectomy, ovariectomized group, and rice root extract of Preparation 1 at a concentration of 50 mg / kg each (OVX + REOS) and 50 mg / kg of non-glycosylated soy isoflavone (OVX + ISF) as a positive control.

Weights were measured weekly for 12 weeks, and the femur of ovariectomized rats sacrificed at the end of the experiment was removed and bone mineral density (BMD) was measured using a dual energy X-ray bone density meter (Norland pDEXA).

The femur extracted from ovariectomized rats was rapidly frozen with liquid nitrogen and then pulverized and used for the test. Total RNA extraction was performed with TRIzol (Invitrogen). 1 ug of the separated RNA was synthesized by adding random primer, dNTP, and PrimeScript ™ Reverse Transcriptase (TaKaRa). The synthesized cDNA was subjected to realtime PCR with SYBR Premix Ex Taq (TaKaRa) using primers to confirm the mRNA expression levels of ALP, Osterix and Runx2, the differentiation factors involved in osteoblast formation.

(2) Weight measurement

All the ovariectomized groups showed a tendency to gain weight, but no significant difference compared to the sham without ovariectomy (see FIG. 12).

(3) Measurement of BMD

(BMD) of the ovariectomized experimental group (OVX) was significantly lower than that of the ovariectomized experimental group (Sham), and compared to the positive control group, non-glycosylated soy isoflavone (ISOFLAVONE) It was confirmed that the rice root extract significantly increased the bone mineral density (see FIG. 13).

(5) Measurement of osteoblast differentiation factor expression level

ALP, Osterix and Runx2, which are related to osteoblast formation from the femur, were significantly reduced in the ovariectomized group (OVX) compared to the ovariectomized group (Sham) The administration of rice root extract significantly increased it again. The rice root extract of Preparation Example 1 showed a significantly higher expression level, especially for Runx2 than the positive control (see Fig. 14).

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Preparation Example 1. Preparation of powder

20 mg of the roots extract of Preparation Example 1

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation Example 2. Preparation of tablets

10 mg of the roots extract of Preparation Example 1

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation Example 3. Preparation of capsules

10 mg of the roots extract of Preparation Example 1

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation Example 4. Preparation of injection

10 mg of the roots extract of Preparation Example 1

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation Example 5. Preparation of a liquid preparation

20 mg of the roots extract of Preparation Example 1

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Preparation Example 6. Preparation of Health Functional Foods

1,000 mg of the rice seed extract of Preparation Example 1

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health functional foods according to the usual methods.

Formulation Example 7. Preparation of Health Functional Food Drink

1,000 mg of the rice seed extract of Preparation Example 1

Citric acid 1,000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional method for producing a healthy functional food drink, and the mixture was stirred and heated at 85 DEG C for about 1 hour. Then, the solution was filtered to obtain a sterilized 2 liter container, sealed sterilized and refrigerated It is used for the production of the health functional food beverage composition of the present invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (15)

A health functional food for improving or preventing bone metabolic diseases containing rice extract as an active ingredient,
Wherein the rice extract comprises at least one compound selected from the group consisting of the following chemical formulas (1), (2) and (3):
[Chemical Formula 1]

(2)

(3)

.
The health functional food for improving or preventing bone metabolic diseases according to claim 1, which promotes osteoblast differentiation and inhibits adipocyte differentiation.
The health functional food for improving or preventing bone metabolic diseases according to claim 2, characterized in that obesity and bone metabolic diseases are simultaneously prevented or ameliorated.
[Claim 2] The health food according to claim 1, wherein the rice extract is rice seed extract, stem or root extract.
The health functional food for improving or preventing bone metabolic diseases according to claim 4, wherein the rice extract is a root extract of a rice harvesting machine.
[Claim 2] The health food according to claim 1, wherein the rice extract is a fraction obtained by fractionating water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
delete A pharmaceutical composition for the prevention or treatment of bone metabolic diseases containing rice extract as an active ingredient,
Wherein the rice extract comprises at least one compound selected from the group consisting of compounds represented by the following formulas (1), (2) and (3):
[Chemical Formula 1]

(2)

(3)

.
The pharmaceutical composition for preventing or treating bone metabolic diseases according to claim 8, which promotes osteoblast differentiation and inhibits adipocyte differentiation.
The pharmaceutical composition for preventing or treating bone metabolic diseases according to claim 9, characterized in that obesity and bone metabolic diseases are simultaneously prevented or treated.
[Claim 9] The pharmaceutical composition according to claim 8, wherein the rice extract is an outpost, stem or root extract of rice.
12. The pharmaceutical composition for preventing or treating bone metabolic diseases according to claim 11, wherein the rice extract is a root extract of a rice harvesting machine.
[Claim 9] The pharmaceutical composition for preventing or treating bone metabolic diseases according to claim 8, wherein the rice extract is water, an organic solvent, or a fraction thereof.
14. The method of claim 13, wherein the organic solvent is at least one solvent selected from the group consisting of alcohols having 1 to 4 carbon atoms, hexane, dichloromethane, ethyl acetate, acetone, chloroform and diethyl ether. A pharmaceutical composition for therapeutic use. delete

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