KR20170069413A - Composition comprising extract of germinated black rice as an effective component for prevention or treatment of obesity or of metabolic bone disease - Google Patents

Abstract

The present invention relates to a composition for preventing or treating obesity or bone metabolism diseases, which comprises germinated brown rice extract as an active ingredient. The germination brown rice extract of the present invention inhibits the expression of aP2, CD36 and PPARγ, genes involved in differentiation of adipocytes, (BMD) and bone strength in an ovariectomized osteoporosis animal model, and is useful for bone metabolic diseases such as obesity and osteoporosis, osteoporosis, or fracture, by promoting the expression of ALP, osterix and Runx2, It can be used as medicine or health functional food.

Description

[0001] The present invention relates to a composition for preventing or treating obesity or bone metabolism diseases, which comprises germinated black rice extract as an active ingredient,

The present invention relates to a composition for preventing or treating obesity or bone metabolism diseases, which comprises germinated black rice extract as an active ingredient. The composition of the present invention is useful as a health functional food for preventing and treating bone metabolism such as obesity or osteoporosis, And in the manufacture of pharmaceuticals.

Osteoporosis refers to a skeletal disorder in which the risk of fracture and bone fragility are increased due to low bone mass and bone destruction. Osteoporosis is a particularly frequent occurrence in postmenopausal women, a disease in which the amount of bone is markedly reduced by the decreased secretion of estrogen. The decrease in the amount of bone may be due to individual differences or various other causes, but if the amount of bone excessively decreases and falls below a predetermined value, fracture easily occurs even in a small impact. 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).

On the other hand, black rice (Oryza sative L) is native to Thailand and has been cultivated in Jindo since 1991 in Korea. Black rice contains a large amount of selenium, and it has cancer prevention effect. There is a recent report in China that 40 patients with rectal cancer were treated with black rice for a prolonged period of time and the condition improved.

In 1998, professor Hwang Tae-Ik of the agriculture department of Chonnam National University also reported that the black rice has excellent efficacy in cancer cell removal and gastric ulcer treatment. In addition, vitamin B and vitamin E in black rice contains more than four times as much as ordinary rice, and it is effective for prevention of diabetes and adult diseases as well as female beauty.

As such, various studies have been conducted in the field of disease treatment or health functional food using black rice.

However, there is a growing interest in germinated grains as a health food, but there is little research on its efficacy in germinated black rice.

Korea Patent No. 626,850 Korean Patent Publication No. 2015-0010671

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and an object of the present invention is to provide a composition for preventing or treating obesity or bone metabolic diseases derived from germinated brown rice, and to produce a health functional food or a pharmaceutical product therefrom.

One aspect of the present invention is to provide a composition for preventing or treating obesity or bone metabolism diseases containing germinated black rice extract as an active ingredient.

Another aspect of the present invention is to provide a health functional food for preventing or ameliorating obesity or bone metabolism diseases containing germinated black rice extract as an active ingredient.

The germinated black rice extract of the present invention inhibits the expression of aP2, CD36 and PPAR.gamma., Genes involved in differentiation of adipocytes, promotes the expression of ALP, osterix and Runx2, genes involved in osteoblast differentiation, and promotes ovariectomized osteoporosis animal model (BMD) and bone strength, and can be used as medicines or health functional foods useful for bone metabolism diseases such as obesity and osteoporosis, osteoporosis or fracture.

In addition, the germinated black rice extract of the present invention is superior in extract yield, adipocyte differentiation inhibiting activity and osteoblast differentiation promoting activity to black rice extract, and further enhances bone density and bone strength in an ovariectomized osteoporosis animal model, It is possible to provide a medicine or a health functional food having a more excellent effect on bone metabolic diseases.

1 is a graph showing an effect of inhibiting adipocyte differentiation of germinated brown rice water extracts according to germination period according to Experimental Example 2-1 of the present invention.
FIG. 2 is a graph showing relative mRNA expression levels of adipocyte-forming differentiation factors (aP2, CD36 and PPARγ) by germinated black rice water extract according to germination period according to Experimental Example 2-2 of the present invention.
FIG. 3 shows the result of ALP staining for confirming osteoblast differentiation promotion effect of germinated black rice water extract according to germination period according to Experimental Example 3-1 of the present invention.
FIG. 4 is a graph illustrating numerical results of the ALP staining shown in FIG. 3 by programming.
FIG. 5 is a graph showing relative mRNA expression levels of osteoblast-related differentiation factors (ALP, Osterix, and Runx2) by germinated black rice water extracts according to germination period according to Experimental Example 3-2 of the present invention.
6 is a graph showing an effect of inhibiting adipocyte differentiation of germinated black rice water extract according to the type of LED light source according to Experimental Example 4-1 of the present invention.
FIG. 7 is a graph showing relative mRNA expression levels of adipocyte-forming differentiation factors (aP2, CD36 and PPARγ) by germinated black rice water extract according to the LED light source type according to Experimental Example 4-2 of the present invention.
FIG. 8 shows results of ALP staining for confirming osteoblast differentiation promotion effect of germinated black rice water extract according to the LED light source type according to Experimental Example 5-1 of the present invention.
FIG. 9 is a graph obtained by digitizing the result of ALP staining shown in FIG. 8 by programming.
10 is a graph showing relative mRNA expression levels of osteoblast-related differentiation factors (ALP, Osterix, and Runx2) by germinated black rice water extract according to the LED light source type according to Experimental Example 5-2 of the present invention.
FIG. 11 is a graph showing the results of the ovariectomy (OVX), the ovariectomy (OVX), the ovariectomy (OVX + BRW) and the ovariectomy (OVX + 3 GBRW) treated with germinated brown rice water extract of germination day 3 of Preparation Example 1 according to the rearing period. 11, * is P < 0.05, ** is P < 0.005, *** is P <
FIG. 12 is a graph showing the bone mineral density of each experimental group when the rat was 19 weeks old in Experimental Example 6. FIG. In Fig. 12, * is P < 0.05, ** is P < 0.005, *** is P <
FIG. 13 is a graph showing the bone strength of each experimental group when the rat became 19 weeks old in Experimental Example 6. FIG. In Fig. 13, * is P < 0.05, ** is P < 0.005, *** is P <

Hereinafter, the present invention will be described in more detail.

The composition for preventing or treating bone metabolic diseases according to the present invention contains germinated black rice extract as an active ingredient.

As used herein, the term &quot; comprising as an active ingredient &quot; means an amount sufficient to achieve the efficacy or activity of the black rice extract.

The bone metabolic diseases of the present invention include osteoporosis, bone formation disorder or fracture.

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

In the present invention, 'osteodystrophy' includes osteomalacia, osteitis fibrosa and the like.

In the present invention, 'fracture' refers to a state in which the continuity of bone or cartilage is completely or incompletely lost or a linear deformation occurs. Fractures are pathologic fractures caused by osteoporosis and tumor osteomyelitis, which are anatomical location, extent of fracture, direction of fracture, presence of open window, number of fractures, stability, dislocation of fracture, And fatigue fractures caused by being applied, and the term "fracture" includes all of them.

The extract of the present invention may be an extract of water containing purified water, an organic solvent having 1 to 4 carbon atoms, or a mixed solvent thereof. In addition, the organic solvent having 1 to 4 carbon atoms may contain a polar solvent such as acetic acid, dimethyl-formamide (DMFO) or dimethyl sulfoxide (DMSO), acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, , 4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, 1- chloropentane, o-xylene, diisopropyl ether, 2- chloropropane, Non-polar solvents such as 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, 1,2-dichloroethane, anniline, 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, the present invention can be applied not only to water, an organic solvent having 1 to 4 carbon atoms, a nonpolar solvent or a mixed solvent thereof, but also a fraction obtained by fractionation with the above solvents or an ultrafiltration membrane having various molecular weights or a constant molecular weight cut- And fractions obtained.

The method for producing the extract of the present invention can be carried out by, for example, drying and finishing germinated brown rice, and then adding water, which is 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 germinated black rice used in the extraction of germinated black rice of the present invention may be germinated for 3 days or more. If the germination period is less than 3 days, the extraction yield and the activity of inhibiting adipocyte differentiation may be lowered. In addition, since the osteoblast differentiation increasing activity is improved as the germination period is increased, the germination period can be 3 days or more, preferably 3 days or more, in consideration of the extraction yield, the adipocyte differentiation inhibiting activity, Germinated black rice germinated for 6 days can be used.

In addition, the germinated black rice used in the extraction of germinated black rice according to the present invention may be a LED (Light Emitting Diode) light source, preferably a VLED (Visible Light Emitting Diode) light source of a visible light region and a white light source ). &Lt; / RTI &gt;

The LED light sources can be classified into light sources of various colors as shown in Table 1 according to wavelengths. By mixing two or more colors selected from red, green and blue, which are the basic light source colors of LEDs, Can be implemented.

The wavelength (λ) LED light source color 620 < / = 780 nm Red (Red) 590 < / = 620 nm Orange (Orange) 570 < / = 590 nm Yellow 490 <?? 570 nm Green 450 < / = 490 nm Blue 420 <?? 450 nm Violet 380 <?? 420 nm Purple 5000 <?? 7000 K White (white)

The germinated black rice extract obtained by using germinated black rice obtained from the dark room shows excellent extraction yield, adipocyte differentiation inhibiting activity and osteoblast differentiation increasing activity. However, in case of germinated black rice extract obtained from germinated black rice germinated under LED light source, It is possible to demonstrate the same level of inhibition of adipocyte differentiation compared to germinated black rice extract obtained from germinated black rice germinated in the dark room, and further improved extraction yield and osteoblast differentiation increasing activity.

The germinated black rice extract obtained by using the germinated black rice germinated under the LED light source may have an increased extraction yield compared to the germinated black germ extract obtained using the germinated black rice germinated in the dark room. Here, the LED light source may include an orange of 590 <? 620 nm, a yellow light source of 570 <? 590 nm, a green light source of 490 <? 570 nm, a light source of 450 < A blue light source of? 490 nm wavelength, a violet light source of 420?? 450 nm wavelength, and a purple light source of 380?? 420 nm wavelength band.

In particular, the germinated black rice extract obtained from germinated black rice germinated under the light sources of Blue, Purple and Mix among the LED light sources exhibits an equivalent level of inhibition of adipocyte differentiation compared to the germinated black rice extract germinated in the dark room.

Among the LED light sources, the germinated black rice extract obtained from germinated black rice germinated under the orange, green and blue light sources exhibits enhanced osteoblast differentiation activity compared to the germinated black rice extract germinated in the dark room.

Accordingly, among the LED light sources, the germinated black rice extract obtained from germinated black rice germinated under a blue light source having a wavelength of 450?? 490 nm can exhibit excellent extraction yield, adipocyte differentiation inhibiting activity and osteoblast differentiation increasing activity.

The composition for preventing or treating obesity or bone metabolism diseases comprising germinated black rice extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions.

The composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Can be used.

Examples of carriers, excipients and diluents that can be included in the composition containing the germinated black rice extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. 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, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the germinated black rice extract of the present invention varies depending on the condition and body weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by the therapist. However, for the desired effect, the extract of the present invention is preferably administered at a dose of 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention provides a health functional food for preventing or ameliorating obesity or bone metabolic diseases, which comprises an extract of germinated brown rice showing an activity of inhibiting adipocyte differentiation, an osteoblast differentiation increasing activity and a bone mineral density increasing activity, and a pharmaceutically acceptable food supplementary 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.

Since the germination black rice extract of the present invention has little toxicity and side effects, it can be safely used for prolonged use even for prophylactic purposes.

When the germinated black rice extract of the present invention is added to a food or beverage for the purpose of preventing or improving obesity or bone metabolic diseases, the amount of the extract in the food or beverage is generally from 0.01 to 15 wt% %, And the health beverage composition can be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient, as long as it contains the extract as an essential ingredient at the indicated ratio, and there is no particular limitation to the liquid ingredient. 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. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to 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.

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

Significant differences between the experimental groups were tested at the p <0.05 level using Analysis of Variance (ANOVA) and Duncan's multiple range test. The same characters in the results of the experimental group showed no statistical significance and there was a significant difference between the different characters.

Production Example 1: Preparation of black rice and germinated brown rice water extract (darkroom germination)

The black rice used in the present invention was harvested directly from the National Institute of Food Science and Technology, Milyang Rural Development Administration, Gyeongsangnam-do. The harvested black rice was soaked in a 30-fold diluted sodium hypochlorite solution at 30 ° C for 24 hours and washed with water. Then, it was spread evenly on the base plate, filled with water at a height of about 1/2 to 2/3 of the Japanese black rice, germinated and germinated in the dark room at a temperature of 30 ° C.

Germinated black rice was obtained in 1 day or 2 days during the germination period, and germinated black rice water extract was prepared for each germinated black rice in the following manner. The germination period was a total of 6 days and germinated black rice was obtained at 0 day, 1st day, 2nd day, 3rd day, 4th day and 6th day respectively, and 0th day means black rice before germination.

The black rice and the sprouted black rice were completely dried, and finely pulverized to obtain a powdery sample.

Water was added to the powder sample 20 times by weight, and the mixture was heated at 30 占 폚 for 24 hours to obtain a black rice water extract and a germinated black rice water extract.

Production Example 2: Production of black rice and germinated brown rice ethanol extract (darkroom germination)

The same powder sample as in Production Example 1 was prepared.

400 ml of a 70% by weight aqueous ethanol solution was added to 20 g of the powder sample, and the mixture was firstly extracted at 120 rpm and 60 ° C. for 24 hours in a shaking incubator. The supernatant was filtered with a Whatman No.1 filter paper, 400 ml of a 70% by weight aqueous ethanol solution was added per 20 g of the sample, and the mixture was extracted again for 24 hours and filtered in the same manner as described above. The filtrate was concentrated under reduced pressure at 45 ° C with a rotary vacuum evaporator and the solvent was completely blown off to obtain a 70 wt% ethanol extract of black rice and a 70 wt% ethanol extract of germinated black rice.

Comparative Preparation Example 1 Preparation of black rice and germinated black rice acetone extract (darkroom germination)

Using acetone as an extraction solvent, black rice and germinated black rice acetone extracts were prepared in the same manner as in Production Example 2.

Experimental Example 1: Germination period and extraction yield of germinated black rice according to extraction solvent

The extraction yields of the water extract and 70% ethanol extract of germinated black rice germinated in the dark and dark rooms prepared in Preparation Examples 1 and 2 were measured and are shown in Table 2.

Germination period Extraction yield (%) Production Example 1 Production Example 2 Comparative Preparation Example 1 Water extract 70% by weight ethanol extract Acetone extract 0 day 1.69 ± 0.21 3.51 + - 0.17 2.42 ± 0.15 Day 1 5.11 + - 0.37 3.49 ± 0.56 2.69 ± 0.13 Day 2 21.86 +/- 1.47 6.33 ± 0.65 3.51 + - 0.11 Day 3 31.20 ± 2.06 12.20 ± 0.27 6.05 ± 0.21 Day 4 17.79 ± 0.94 13.87 ± 0.69 6.98 + 0.17 Day 6 9.83 + - 0.59 12.79 + - 0.93 5.99 + - 0.43

As shown in Table 2, the extraction yield of germinated black rice was higher than that of the black extract before germination (0 day difference), the water extract and the 70 wt% Ethanol extracts showed higher germination yield.

The water extract and the 70 wt% ethanol extract were found to have an excellent extraction yield, and the water extract and the 70 wt% ethanol extract had the same yields. However, the extraction yield of the water extract was somewhat higher appear.

Therefore, the water extract of Preparation Example 1 having a high extraction yield from the extracts was used to test the effect of germination time on the differentiation of adipocytes and the effect of osteoclast differentiation.

Experimental Example 2: Inhibition effect of germination water on the differentiation of adipocyte according to the duration of black rice and germination

2-1. Inhibition of adipocyte differentiation in C3H10T1 / 2 cells

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 and adipocytes.

The C3H10T1 / 2 cell line was cultured in adipocyte differentiation medium containing 1 uM dexamethasone, 5 ug / ml insulin and 20 nM PPARγ for adipocyte differentiation at a concentration of 2.5 × 10 4 / ml.

The adipocyte differentiation medium was cultured for 10 days at a rate of 20 ug / ml for each germination period of germination period according to the germination period of the preparation example 1, and then the inhibition effect on the adipocyte differentiation by the germination period was measured. Respectively. At this time, the addition of DMSO alone without addition of the germinated black rice water extract according to germination period of Preparation Example 1 was used as a negative control (Control).

In FIG. 1, it was found that the water extract of germinated black rice having a germination period of 3 days or more improved the effect of inhibiting adipocyte differentiation compared to the water extract of black rice (day 0) before germination.

2-2. Analysis of adipocyte differentiation factor expression through real-time RT-PCR

The C3H10T1 / 2 cell line was treated with the germination medium for germination period of 9 days for the germination period of the adipocyte differentiation medium and the germination period of the preparation example 1, and then aP2, CD36 and PPARγ The relative mRNA expression level of the mRNA was determined and shown in Fig.

2, the expression level of adipocyte differentiation factor was decreased compared to the water extract of germinated black rice before germination (0th day) in germinated black rice with germination period of 3 days or more, and the same tendency as the inhibition of adipocyte differentiation shown in Fig. 1 .

Experimental Example 3: Promoting osteoblast differentiation promoting effect of germinated brown rice water extracts on black rice and germination period

3-1. ALP (alkaline phosphatase) staining

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 and adipocytes.

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 for 3 days in a 6-well plate with a medium containing 10 mM glycerophosphate and 50 μg / ml ascorbic acid for osteoblast differentiation at a concentration of 2.5 × 10 4 / ml. Of germinated brown rice water extracts were added at a concentration of 20 ㎍ / ml, and the medium was changed every 3 days for 6 days.

The germination period of the germination period of Preparation Example 1 and the addition of distilled water (DW) alone were used as a negative control. The germination period of germination period according to germination period of ALP Dyeing results for activity confirmation and numerical values obtained by programming the result of the dyeing are shown in FIG. 3 and FIG. 4, respectively.

In the case of lipid differentiation, the stained cells could be dissolved, and the absorbance was measured to obtain the result. In the case of ALP staining, the plate was scanned through a scanner, and the result was calculated as Lab value to obtain FIG. 4 (Lab: L * a * b * In L * a * b * color space, L * indicates brightness, L * = 0 means black, L * = 100 means white, a * indicates either red or green B * is yellow and blue, b * is negative if b * is positive, and yellow if b * is positive.) If a * is negative, it is a green color. ALP staining is indicated by the -b value in the Lab color value because it is blue when the bone differentiation activity is higher.

3 and 4, it was confirmed that ALP activity of the germinated black rice extract was increased as the germination period was increased.

3-2. Analysis of osteoblast differentiation factor expression through real-time RT-PCR

5 shows the mRNA expression levels of ALP, Osterix and Runx2, which are the differentiation factors involved in osteoblast formation, by real-time PCR after differentiating C3H10T1 / 2 cells for 3 days from the above 3-1 .

FIG. 5 shows that the mRNA expression levels of ALP, osterix and Runx2 in the group treated with germinated black rice water extract were significantly increased compared to the group treated with black water extract. As the germination period was increased, the amount of mRNA expression of ALP, osterix and Runx2 was increased in the groups treated with the germinated black rice water extract, and there was no significant difference in germination date, indicating the same tendency as in FIGS. 3 and 4 .

In the Experimental Example 2 and Experimental Example 3, the effect of the germinated brown rice water extract on germination day 3 was excellent in the adipocyte differentiation inhibition effect and the osteocyte division promoting effect test. As a result, , An experiment was conducted to optimize the condition of germinated brown rice.

Production Example 3: Preparation of germinated black rice water extracts according to LED light sources

Germinated brown rice water extract was prepared by the same method as in Production Example 1 except that the germinated brown rice water extract was prepared under an LED light source instead of the dark room and the extraction yields thereof are shown in Table 3. [ At this time, the extraction yield of germinated black rice water extract according to the type of LED light source was measured by different kinds of LED light sources, and the dark germination group of Preparation Example 1 was used as a control group.

division LED light source type Extraction yield (%) Control group
Production Example 1 (Germination Day 3) Dark (Dark) 31.20 ± 2.06 Production example 3-1 Red 36.89 ± 1.29 Production example 3-2 Orange 40.59 ± 2.99 Production Example 3-3 Yellow 32.84 + - 4.38 Production example 3-4 Green 31.98 ± 2.24 Production Example 3-5 Blue 39.63 + - 0.70 Production Example 3-6 Navy 36.08 ± 2.22 Production Example 3-7 Purple 39.13 + - 3.34 Production Example 3-8 Mix 38.09 ± 1.13

As shown in Table 2, the extraction yield of germinated black rice water extract was improved when germinated under an LED light source, compared to when germinated black rice was germinated in a dark room.

Among the LED light sources, the extraction yield of germinated black rice water extracts obtained under the orange, blue, purple and mix (white) light sources was high.

Experimental Example 4: Inhibitory effect of germinated black rice water extract on adipocyte differentiation depending on the type of LED light source

4-1. Inhibition of adipocyte differentiation in C3H10T1 / 2 cells

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 and adipocytes.

The C3H10T1 / 2 cell line was cultured in adipocyte differentiation medium containing 1 uM dexamethasone, 5 ug / ml insulin and 20 nM PPARγ for adipocyte differentiation at a concentration of 2.5 × 10 4 / ml.

The germinated brown rice water extracts of the germinated third day of germination according to the LED light source of Preparation Example 3 were added to the adipocyte differentiation medium in an amount of 20 ug / ml each for 9 days and then the germinated brown rice water extract The adipocyte differentiation inhibitory effect was measured and the results are shown in Fig. At this time, the addition of distilled water (DW) alone to the germinated black rice water extract of germination day 3 according to the LED light source of Preparation Example 3 was used as a negative control.

In FIG. 6, germinated black rice water extracts obtained from Blue, Purple and Mix light sources of LED light sources showed the same level of fat cell degradation as germinated black rice water extracts obtained from Dark, It can be seen that the fat cell degradation effect of germinated brown rice water extract is not increased.

4-2. realtime RT - PCR (Fig. adipocyte Analysis of differentiation factor expression

The C3H10T1 / 2 cell line was treated with the adipocyte differentiation medium and germinated brown rice water extract of germination day 3 according to the LED light source of Preparation Example 3 for 9 days, and then real-time RT-PCR was performed to determine important factors of adipocyte formation The relative mRNA expression levels of aP2, CD36, and PPARy were confirmed and shown in FIG.

In FIG. 7, the germinated brown rice water extract obtained under the blue, purple and mixed light sources of the LED light source showed the same level of fat cell degradation as the germinated brown rice water extract obtained from the dark room. The same tendency as that of cell differentiation inhibition was shown.

From this, it can be seen that the degradation effect of the germinated black rice water extract is not increased by the LED light source.

EXPERIMENTAL EXAMPLE 5: Promoting osteoblast differentiation promoting effect of germinated black rice water extract according to kinds of LED light sources

5-1. ALP (alkaline phosphatase) staining

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 and adipocytes.

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 for 3 days in a 6-well plate with a medium containing 10 mM glycerophosphate and 50 μg / ml ascorbic acid for osteoblast differentiation at a concentration of 2.5 × 10 4 / ml. Of germinated brown rice water extracts were added at a concentration of 20 ㎍ / ml, and the medium was changed every 3 days for 6 days.

The addition of distilled water (DW) without addition of the germinated black rice water extract of the germinated third day of germination according to the LED light source of Preparation Example 3 was used as a negative control, and germination according to the LED light source of Preparation Example 3 Dyeing results for confirming ALP activity according to the kinds of LEDs of germinated black rice water extract on day 3, and graphs obtained by digitizing the result of the dyeing by programming are shown in FIG. 8 and FIG. 9, respectively.

8 and 9, it was confirmed that the germinated brown rice water extract of germination day 3 of germination obtained under the LED light source increased ALP activity compared to germinated brown rice water extract of germination day 3 obtained in darkness as a whole. In addition, it can be seen that the level of increase of ALP activity by the germinated black rice extract of germination day 3 obtained under the orange, green and blue light sources of the LED light source is high.

5-2. Analysis of osteoblast differentiation factor expression through real-time RT-PCR

In the above 5-1, C3H10T1 / 2 cells were differentiated for 6 days using the germination brown rice extract of germination day 3 obtained under the light sources of Orange, Green and Blue, The mRNA expression levels of ALP, Osterix and Runx2, which are the differentiation factors involved in osteoblast formation, were confirmed by -time PCR and shown in FIG.

In FIG. 10, the mRNA expression levels of ALP, Osterix and Runx2 in the group treated with germinated black rice extract of germination day 3, which were obtained under the orange, green and blue light sources, were significantly increased compared to the germination black rice extract of germination third day .

Experimental Example 6: Ovariectomy of germinated black rice water extract (ovariectomy) Test using an animal model of osteoporosis

To examine the effect of the water extract of black rice (BRW) of Preparation Example 1 and germinated black rice water extract (3 GBRW) of germination day 3 on the treatment and prevention of osteoporosis, the water extracts were respectively administered to ovariectomized rats, Bone mineral density was measured and histological analysis was performed.

6-1. Animal breeding and ovariectomy

Ten-week-old female SD rats were purchased from Orient Biotech and had a purifying period of one week. At 11 weeks of age, ovariectomies were performed and there was a one week recovery period. Experimental animals were housed in a single cage. The environmental conditions were adjusted to room temperature 24 ± 2 ℃, relative humidity 55 ± 10%, and illumination time 12 hours. Feed and water were freely available.

Experimental animals were divided into three groups. Ten experimental ovariectomized group (SHAM), 10 ovariectomized group (OVX) and 10 ovariectomized group were treated with lateral resection. (OVX + 3 GBRW 100 mg / kg) administered with the germinated brown rice water extract (3 GBRW) of the germination day 3 germination of the preparation example 1 after ovariectomy were divided into 10 experimental groups (OVX + BRW 100 mg / kg) . Ovariectomy was performed by using anesthesia with Zoletil and rumpun, and then depilating and incising the skin, removing the ovaries and suturing. The test samples were orally administered for a total of 8 weeks.

As a result, the ovariectomy group (OVX) showed a tendency to increase in body weight compared to the ovariectomized group (SHAM) due to the decrease of oestrogen secretion due to ovariectomy, and the brown rice water extract (OVX + BRW 100 mg / kg) and the control group (OVX + 3 GBRW) administered with the germinated brown rice water extract of the germination day 3 of Preparation Example 1 after ovariectomy were compared with the ovariectomized group (OVX) But a significant difference was not observed (FIG. 11).

6-2. BMD measurement

When the animal was 19 weeks of age, the femur and tibia were separated and the femur was used to measure bone density. BMD was measured using pDEXA ^® (Forearm: X-Ray, NORLAND, Bone Densitometer, USA).

As a result, the BMD of the ovariectomized group (OVX) was significantly lower than that of the ovariectomized group (SHAM), and the ovariectomized BMD of the experimental group (OVX) (OVX + 3 GBRW) of the germinated brown rice water extract of germination day 3 of Preparation Example 1 increased significantly compared to the ovariectomized group (OVX) of the ovariectomized group (OVX + BRW) (BMD) was higher than that of the experimental group (OVX + BRW) to which the black rice water extract of Preparation Example 1 was administered (FIG. 12).

6-3. Bone Strength (BS) Measurement

(OVx + BRW) in which the black water extract of Preparation Example 1 was administered (Ovx + BRW) after the ovariectomy, and the germination 3 of the preparation example 1 after the ovariectomy The right femur was extracted from each individual of the experimental group (Ovx + 3 GBRW) treated with the germination water of the first germination, and then the bone strength, ie, breaking force (kg), was measured.

As a result, the bone strength (BS) of the ovariectomized group (OVX) was not significantly different from that of the ovariectomized group (SHAM) but it was decreased to some extent. After ovariectomy, the brown rice water extract of Preparation Example 1 was administered The bone strength (BS) of one experimental group (Ovx + BRW) was higher than that of ovariectomized group (OVX), but there was no significant difference. Also, in the experimental group (OVX + 3 GBRW) showed a significant increase in bone strength as compared to the experimental group (OVX + BRW) to which the black rice water extract of Preparation Example 1 was administered (FIG. 13).

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. The germinated brown rice water extract used in Formulation Examples 1 to 7 was the germinated brown rice water extract of germination day 3 obtained from the Blue light source of Preparation Example 3-5.

Preparation Example 1. Preparation of powder

20 mg of the germinated brown rice water extract of Preparation Example 3-5

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 germinated brown rice water extract of Preparation Example 3-5

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 germinated brown rice water extract of Preparation Example 3-5

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 germinated brown rice water extract of Preparation Example 3-5

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 germinated brown rice water extract of Preparation Example 3-5

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.

Formulation Example 6. Preparation of Healthy Foods

1,000 mg of the germinated brown rice water extract of Preparation Example 3-5

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 food compositions according to conventional methods.

Formulation Example 7. Preparation of health drink

1,000 mg of the germinated brown rice water extract of Preparation Example 3-5

Citric acid 1,000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and then refrigerated And then used in the production of the health 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.

It will be apparent to those skilled in the art that the present invention is not limited to the embodiments described above and that various changes and modifications may be made without departing from the spirit and scope of the present invention as defined by the appended claims. As shown in FIG.

It will be understood by those skilled in the art that the present invention may be embodied in many other specific forms without departing from the spirit or scope of the invention as defined by the appended claims and their equivalents. .

Claims (13)

A composition for preventing or treating obesity or bone metabolism diseases, comprising germinated black rice extract as an active ingredient. The composition for preventing or treating obesity or bone metabolic diseases according to claim 1, wherein the bone metabolic disease is osteoporosis, osteogenesis disorder or fracture. The composition for preventing or treating obesity or bone metabolism according to claim 1, wherein the germinated black rice extract is an extract of water, an organic solvent having 1 to 4 carbon atoms, or a mixed solvent thereof. 4. The composition for preventing or treating obesity or bone metabolism disorder according to claim 3, wherein the germinated brown rice extract is germinated brown rice extract extracted with water as a solvent. 4. The composition for preventing or treating obesity or bone metabolism disorder according to claim 3, wherein the germinated black rice extract is a fraction obtained by fractionating the extract with water, an organic solvent having 1 to 4 carbon atoms, or a mixed solvent thereof. [5] The germinated black rice extract according to claim 3, wherein the germinated black rice extract is obtained by stirring, extracting with hot water, extracting with cooling water, refluxing cooling extraction, ultrasonic extraction, or supercritical extraction using an extraction solvent of 1 to 50 times the weight of dried germinated black rice Wherein said composition is for preventing or treating obesity or bone metabolic diseases. The composition for preventing or treating obesity or bone metabolic diseases according to claim 1, wherein the germinated brown rice is germinated for 3 days or more. The composition for preventing or treating obesity or bone metabolic diseases according to claim 7, wherein the germinated black rice is germinated for 3 to 6 days. The composition for preventing or treating obesity or bone metabolic diseases according to claim 1, wherein the germinated black rice is germinated under an LED light source. The composition for preventing or treating obesity or bone metabolism disorder according to claim 9, wherein the light emitting diode (LED) light source is a VLED (Visible Light Emitting Diode) light source in a visible light region and a white light source in which the VLED light sources are mixed. The LED light source according to claim 10, wherein the LED light source comprises an orange of 590 <? 620 nm, a yellow light source of 570 <? 590 nm, a green light source of 490 <? 570 nm, , A blue light source of 450?? 490 nm wavelength, a violet light source of 420?? 450 nm wavelength, and a purple light source of 380?? 420 nm wavelength band. A composition for preventing or treating bone metabolic diseases. 12. The composition for preventing or treating obesity or bone metabolism disorder according to claim 11, wherein the LED light source is a blue light source having a wavelength of 450?? 490 nm. A health functional food for preventing or ameliorating obesity or bone metabolism diseases containing germinated black rice extract as an active ingredient.

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