KR20140061825A - Perillae semen extracts for enhancing differentiation of osteoblast and inhibiting differentiation of osteoclast and use of thereof as bone formation promoting products and bone resorption inhibitory products - Google Patents

Abstract

The present invention relates to a perillae semen extract and a product comprising the same as an active ingredient which can be applied to a pharmaceutical composition, a fermented milk, or a functional health food that can help alleviating elderly bone disease or bone diseases caused by menopause by using the perillae semen extract capable of effectively suppressing functional and differentiation reduction of osteoblast and activity increase of osteoclast which causes bone diseases resulting from imbalance due to increase in bone resorption for bone formation during bone remodeling.

Description

[0001] The present invention relates to a self-extracting substance having an osteoblast differentiation promotion and osteoclast differentiation inhibiting activity, and a product containing the self-extracting substance as an active ingredient. [0003] Perillae semen extracts for enhancing differentiation of osteoblast and osteoclast differentiation products and bone resorption inhibitory products}

The present invention relates to a self-extracting substance having an osteoblast differentiation promotion and an osteoclast differentiation inhibiting activity and a product containing the self-extracting substance as an active ingredient, and more particularly, to a bone remodeling product, The present invention relates to a self-component extract having an activity of effectively promoting osteoblast activation and differentiation and inhibiting osteoclast differentiation from a decrease in bone formation and bone resorption, A pharmaceutical composition for promoting growth and development, a food composition, and the like.

The body supports the human body and is composed of cartilage, which is a tissue that connects 200 different bones and different bones. They provide basic and versatile functions such as protecting important organs, providing a microenvironment for hematopoiesis. It is also an important part of the human body that not only supports the muscles or organs but also stores calcium or other essential minerals in the body, such as phosphorus or magnesium. Thus, adult bones do not stop growing until they die, and they maintain a balanced balance by continuously dynamically and continuously repeating the generation and absorption processes that remove old bones and replace them with new bones. This is called bone remodeling (Yamaguchi A. et al., Tanpakushitsu Kakusan Koso., 50 (6Suppl), pp.664-669, 2005). The turnover, which removes the old bone and replaces it with the new bone, is essential for restoring the fine damage of the bone caused by growth and stress and maintaining proper bone function (Cohen-Solal M. et al., Therapie., 58 (5), pp. 391-393, 2003).

It is known that two types of cells are involved in aggregate formation. One of the cells is osteoblast that produces bone, and the other is osteoclast that destroys bone. These cells are derived from different cells, and mesenchymal stem cells are differentiated into cells of osteoblast, myoblast, adipocyte, fibroblast, chondrocyte, etc. And hematopoietic stem cells are differentiated into osteoclasts, macrophages, T cells, B cells, dentritic cells, basophiles, ), Eosinophile, neutrophile, platelet, red blood cell and the like. Run-2 (Runt-related transcription factor 2), MyoD (myoepithelial cell differentiation factor), PPARγ (peroxisome proliferator-activated receptors γ), and Sox9 the control of inhibiting the expression and induce differentiation (Yamashita S. et al., Exp Cell Res., 315 (13), pp.2231-40, 2009, Charles A. et al., Exp Cell Res., 300 ( Et al. , Dev Cell., 8 (5), pp. 406-17, 2004, David V. et al. , Endocrinology., 148 (5), pp.2553-62, pp.727-38, 2005, Wang H. et al ., J Bone Miner Res., 23 (6), pp.939-48, 2008, Lengner CJ. et al., J Biol Chem., 280 (16) , pp. 15872-9, 2005). In addition, Sox9, which is a major factor of chondrocyte differentiation, inhibits the maturation and differentiation of osteoblasts. Wnt, which is a differentiation factor of osteoblast, promotes Runx2 but inhibits Sox9 by degrading osteoblast. Osterix, inhibit (Dy P. et al, Dev Cell , 13;... 22 (3), pp.597-609, 2012, Gunil I. et al, Journal of the Society of rheumatism, 15 (1), 2008, Tominaga H et al ., J Bone Miner Metab., 27 (1), pp. 36-45, 2009).

Osteoblasts produce RANKL (Receptor activator of NF-κB ligand) and its inducible receptor (decoy receptor), OPG (Osteoprotegerin). When RANKL binds to the receptors RANK (Receptor activator of NF-κB) on the surface of osteoclast progenitor cells, osteoclast precursor cells are maturated into large polynuclear osteoclasts and bone resorption occurs. However, when OPG binds to RANKL, the binding between RANKL and RANK is blocked, resulting in inhibition of osteoclast formation and no more bone resorption than necessary (Theill LE et al ., Annu Rev Immunol., 20, pp. 795-823 , 2002; Wagner EF. Et al ., Curr Opin Genet Dev., 11, pp. 527-532, 2001). The activity of these osteoclasts results in the absorption or destruction of old bone, which is used to maintain bodily function by piercing the bone and releasing a small amount of calcium into the bloodstream (William J. et al., Nature 423, pp. 3737342, 2003).

On the other hand, the osteoblasts generated from bone cells fill holes with collagen and cover the hydroxyapatite of calcium and phosphorus to form new hard bones (Stains JP et al ., Birth Defects Res C Embryo Today. 75 (1), pp. 72-80, 2005). It takes about 100 days for the bones to begin to break down and rebuild into new bones (Schwarz EM. Et al ., Curr Opin Orthop., 11, pp. 329-335, 2000). In infants, bone calcium is 100% changed within a year, but about 10-30% of the bone is reshaped every year in adults, and the osteopenic rate and osteotomy speed are the same so that bone density can be maintained as before.

These two processes, namely, bone resorption and bone formation, are closely linked to each other and are important for maintaining the normal structure of the bone. On the other hand, osteoporosis, which is becoming a big problem in aging society, is a disease in which bone mass is decreased due to various causes and the risk of fracture is continuously increased due to degeneration of microstructure of bone tissue. (Iqbal MM., South Med. J., 93 (1), pp. 2-18, 1999). In addition, 2000). Inside the normal bone is a dense structure like a net, but in the case of osteoporosis, the gap between the structures is widened and the microstructure is thinned and weakened, so that the bone is easily broken even in a small impact (Stepan JJ et al , Endocr Regul., 37 (4), pp. 225-238, 2003). Postmenopausal osteoporosis (postmenopausal osteoporosis), which is characterized by rapid bone loss (2-3% per year) at the onset of menopause and slowly increasing risk of fracture of the spine Senile osteoporosis, which causes progressive bone loss in the hip bone and vertebrae, and age-related diseases (endocrine disease, gastrointestinal diseases, malignant tumors) or drugs Secondary osteoporosis resulting from alcoholism, smoking, and accident (Rosen CJ, N., et al.) Are classified into two types: osteoporosis (corticosteroids, chemotherapy, thyroid hormones, anticonvulsants, anticoagulants, methotexate, cyclosporine and GnRH) Engl J Med., 353 (6), pp. 595-603, 2005; Davidson M., Clinicain Reviews., 12 (4), pp.75-82, 2002).

Currently, medicines used for the prevention and treatment of osteoporosis mostly inhibit bone resorption, so that it is impossible to completely recover the already advanced bone loss, and thus it is a reality that the ultimate goal of osteoporosis can not be completely prevented. However, there is a need for a scientific approach to osteoporosis prevention and treatment, and the effect of osteogenesis on bone regeneration (Chp SH et al ., Korean J Obstet Gynecol. 39: 1497- 1506, 1996, Boonene et al ., J Internal Medicine., 242: 285-290, 1997)

Periodontal disease can be divided into gingivitis and periodontal disease according to the severity of the disease. Bacterial inflammation is called gingivitis when it progresses to the periphery of the gums and gums. The more periodontal disease, the deeper the depth of the periodontal pouch, the periodontal ligament becomes deeper, the periodontal ligament is inflamed, and the bone loss is periodontal disease. To heal periodontal tissue, it is necessary to regenerate periodontal ligament connecting tooth and alveolar bone. It is known that proliferation of osteoblast is most important in this healing process. Periodontal ligament cells have similar characteristics to osteoblasts and differentiate to form hard tissues. Currently, the extract of Zea Mays L. is the only drug derived from natural products developed as a treatment for periodontal disease in Korea and abroad. It is commercially available under the trade name INVENDOL, but its pharmacological mechanism has not been elucidated. There has been no development as a drug showing excellent effects on periodontal disease. In particular, there has been no attempt to treat periodontal disease by increasing osteoblast activity.

Perillae semen (PS) is a fruit of Perilla frutescens L. Britton var. Acuta KUDO. It is near oval or spherical shape and has a diameter of 0.6 ~ 2mm. Outer surface is grayish brown or dark grayish brown with slightly raised net pattern. The donation has a pointed, white dotted fruit stalk. The fruit skin is thin and easily broken when pressed. Seed shells are membranous and cotyledons are rich in oil. There is almost no smell, but if you chew, there is a peculiar smell. The taste is tough and slightly spicy. If you use it as an external medicine, put out the powder and apply it to the foundation. Pharmacological experiments revealed smooth muscle rooting activity, hypotensive action, and excitatory action on the respiratory center, and anticancer activity, antimicrobial activity, whitening effect, and anti-HIV activity were reported.

There is a registered patent (Registration No. 10-0595735) regarding regeneration of chondrocyte of ganghwam, prolene, and lobular mixed extract. However, the effect of prodrug extract on the osteoblast differentiation promoting activity and osteoclast differentiation inhibition effect It has not been disclosed. Chondrocytes and osteoblasts are derived from mesenchymal stem cells and differentiated into different cells such as osteoblasts, myoblasts, adipocytes, fibroblasts, and chondrocytes, and the osteoblast-promoting factor's cartilage cell suppression mechanism and osteoblast- since cell suppression mechanism such as is already known for a different cell (Dy P. et al, Dev cell , 13;... 22 (3), pp.597-609, 2012, Gunil I. et al, Journal of the Society of rheumatism, 15 (1), 2008, Tominaga H. et al ., J Bone Miner Metab., 27 (1), pp. 36-45, 2009).

Accordingly, the inventors of the present invention have found that the extract of Z-element extract has an activity to promote osteoblast differentiation and inhibit osteoclast differentiation in order to identify a physiologically active substance for natural or medicinal use in daily life. Thereby completing the invention.

Korean Patent Registration No. 0595735 (title of the invention: herbal composition for treating cartilage regeneration and osteoarthritis, Jun. 30, 2006)

It is an object of the present invention to provide a pharmaceutical composition, a food composition, a fermented milk, a health functional food, and the like containing a self-component extract having the osteoblast differentiation promotion and osteoclast differentiation inhibiting activity and the self-component extract as an active ingredient .

In order to accomplish the above object, the present invention provides a pharmaceutical composition, a food composition, a fermented milk, a health functional food, a pharmaceutical composition, a cosmetic composition, a cosmetic composition, .

Hereinafter, the present invention will be described in detail.

" Perilla frutescens L. Britton var. Acuta Kudo" as a source of the extract used as an active ingredient of the present invention is an annual herb of Lamiaceae. It is called leaflet, It stands straight and has a height of 20 ~ 80cm. It has a square cross section, mauve light and scent. Leaves are opposite, wide ovate, pointed end, round bottom, with sawtooth on edge. There are hairs on both sides of the leaf, there is long hairs on the top of the back side, and petiole is long. The essential oil constituents of the whole plant are arginine, cumic acid, cyanidin-3- (6 (meth) acrylamide) in addition to a small amount of about 55% of perylaldehyde, 20 to 30% of 1-limonene and a- p-coumaroyl- beta -D-glucoside) -5- [beta] -D-glucoside, and the purified fat component of the leaves contains isoegoma ketone. It is used as sweating, calming, dryness, diuretic, soothing and analgesic.頭 head is an old stem near the roots and roots of the chrysanthemum. It is effective in reducing gout, sweating, and germinating, and is effective in lowering 气, It is effective in treating runny nose (Ji-Sup and Shin Min-gyo;

The extract of the present invention is preferably obtained by extracting from various organs (for example, leaves, stems, roots or seeds) of a garnish, and most preferably, extracted from seeds of a garlic. (A) water, (b) an anhydrous or hydro-lower alcohol having 1 to 4 carbon atoms (e.g., methanol, ethanol, propanol, butanol (D) acetone, (e) ethyl acetate, (f) chloroform, (g) 1, 2-propanol, 3-butylene glycol, (h) hexane, (i) diethyl ether, (j) butyl acetate and the like. Preferably, the self-component extract of the present invention may be water, methanol or ethanol, most preferably ethanol, and particularly preferably 70% ethanol may be used. However, it is not limited thereto.

The child element extract may be a diluent or concentrate of the extract obtained by extraction, a dried product obtained by drying the extract, or a preparation or purified product of the extract.

More specifically, a method of obtaining a magnetic element extract using water is repeatedly performed at a temperature of 100 ° C two times using 5 to 10 times of water as a sample magnetic element. The extract can be prepared by filtration using filter paper at least once, lyophilized and powdered, and used as a sample.

In detail, a method of obtaining a magnetic element extract using a mixed solvent of water and ethanol (70% ethanol) is as follows. A magnetic element as a sample is mixed with 5 to 10 times of a mixed solvent of water and ethanol (70% ethanol) Followed by refluxing twice at 30 to 80 ° C for 2 to 5 hours, and then the extract was added to Watman No. 3. Filtered twice using a filter paper, concentrated using a rotary evaporator, lyophilized and powdered, and used as a sample.

The pharmaceutical composition for preventing and / or treating osteoporosis comprising an extract of a self-component having the osteoblast differentiation promoting activity and osteoclast differentiation inhibiting activity according to the present invention as an active ingredient can be used as a pharmaceutical composition including a pharmaceutically acceptable carrier And may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method.

The bone diseases include osteoporosis, Paget's bone disease, rickets, nephrolithiasis in renal failure patients, rheumatic or degenerative bone disease, bone metastatic lesion, primary bone-derived tumors, periodontal disease, inflammatory alveolar bone An inflammatory disease, an inflammatory disease, an inflammatory disease, an inflammatory disease and an inflammatory bone resorption disease.

The composition for promoting bone strengthening or growth promotion, which contains the self-extracting ingredient having the osteoblast differentiation promotion activity and osteoclast differentiation inhibiting activity as an active ingredient of the present invention, can be provided in the form of fermented milk, functional beverage, , The food composition may include a foodstuff acceptable food supplement additive.

The fermented milk containing the self-extracting ingredient having the activity of promoting osteoblast differentiation and osteoclast differentiation according to the present invention as an active ingredient can be prepared by combining the lactic acid bacteria culture broth, the mixed juice syrup and the self- And then cooled to < RTI ID = 0.0 > 10 C < / RTI >

The health functional food containing the self-extracting ingredient having the activity of promoting differentiation of osteoblasts and inhibiting osteoclast differentiation according to the present invention is characterized in that nutritional supplement components (vitamin B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid ester, nicotinic acid amide) and oligosaccharide are added and mixed in a high-speed rotary mixer. Sterile purified water is added to the mixture, mixed and molded into granules having a diameter of 1 to 2 mm. The molded granules are dried in a vacuum drier at 40 to 50 ° C. and then passed through 12 to 14 mesh to uniformly produce granules. The granules are extruded in appropriate amounts to be purified or powdered or filled into hard capsules, .

The self-component extract of the present invention promotes the differentiation of osteoblasts and inhibits the differentiation of osteoclasts, resulting in an imbalance due to an increase in bone resorption for bone formation in bone remodeling The present invention can be applied to pharmaceutical compositions and functional foods that can improve bone diseases caused by osteopathy of the elderly or menopause of women, for example.

FIG. 1 is a graph showing the effect of the self-extract of the present invention on cytotoxicity of mouse skull osteoblasts.
Fig. 2 is a graph showing the effect of the self-extract of the present invention on the cytotoxicity of the mouse skull osteoblast cell line MC3T3-E1.
FIG. 3 is a graph showing the effect of the self-extract of the present invention on cytotoxicity of human osteoblast cell line HOS cell line.
FIG. 4 is a graph showing the effect of the self-extract of the present invention on the basic phosphatase of the mouse skull osteoblast.
FIG. 5 is a graph showing the effect of the magnetic element 70% ethanol extract of the present invention on the basic phosphatase of the mouse skull osteoblast.
FIG. 6 shows the effect of the self-extract of the present invention on alveolar-red staining of the effect of osteocarcinosis on mouse skull osteoblasts.
FIG. 7 is a graph showing the effect of the magnetic element extract of the present invention on bone calcification of mouse skull osteoblasts measured by absorbance.
FIG. 8 is a photograph showing the inhibitory effect of RANKL induced osteoclast differentiation of the hot-water extract of the magnetic element of the present invention.
FIG. 9 is a photograph showing the effect of inhibition of RANKL-induced osteoclast differentiation by the self-extract of the present invention with a mixed solvent of water and ethanol.
FIG. 10 is a graph showing the number of large polynuclear osteoclasts inhibited by RANKL-induced osteoclast differentiation of the magnetic element hydrothermal extract of the present invention.
FIG. 11 is a graph showing the number of large polynuclear osteoclasts inhibited by RANKL-induced osteoclast differentiation of the 70% ethanol extract of the magnetic element of the present invention.

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

≪ Example 1 >

Production of magnetic element extract

1-1. Production of hot water extract of magnetic element

The process of adding 5 to 10 times of water to the magnetic element and heating it at 100 占 폚 for 2 hours was repeated twice, and the extract was added to Watman No. 3. Filtered twice using filter paper, lyophilized and powdered, and used as a sample.

1-2. Preparation of extract of magnetic element by mixed solvent of water and ethanol (70% ethanol)

(70% ethanol) of 5 to 10 times of water and ethanol was added to the sample, and the mixture was repeatedly extracted twice at 70 to 80 ° C for 2 to 5 hours. The mixture was filtered twice using a filter paper, concentrated using a rotary evaporator (EYELA, NE-1001, Japan), lyophilized and powdered, and used as a sample.

≪ Example 2 >

Of a pharmaceutical composition containing a self-extracting ingredient as an active ingredient

Manufacture of liquid agent

100 mg of lyophilized powder of a hot-water extract of the magnetic element of Example 1 or lyophilized powder of a 70% ethanol extract of a magnetic element, 10 g of isomerized sugar, and 5 g of mannitol were dissolved and dissolved in purified water in accordance with a conventional liquid preparation method After adding proper amount of lemon flavor, purified water was added to adjust the total volume to 100 ml, and the solution was filled in a brown bottle and sterilized to prepare a liquid preparation.

Preparation of capsules

100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed thoroughly with 100 mg of the lyophilized powder or the lyophilized powder of the magnetic element 70% ethanol extract of Example 1, The capsules were filled in hard gelatin capsules according to the manufacturing method.

≪ Example 3 >

Production of fermented milk containing fermented tea extract as an active ingredient

A method for producing a fermented milk containing the present invention's extract as an active ingredient is as follows.

The lactic acid bacteria culture solution was prepared by stirring 95.36 wt% of crude oil and 4.6 wt% of skim milk powder (or mixed powdered milk) and measuring specific gravity at 15 ° C from 1.0473 to 1.0475, a titratable acidity of 0.200 to 0.220%, pH of 6.65 to 6.70, of Brix (Brix ^o) were mixed such that the degree of 16.3 ~ 16.5%. After mixing, the mixture was heat-treated with UHT (sterilized at 135 ° C. for 2 seconds), cooled to 40 ° C., added with 0.02% by weight of Streptococcus thermophilus and Lactolytic enzyme (Valley laboratory, USA) The total number of lactic acid bacteria in the medium was 1.0 × 10 ⁹ cfu / ml or more, the titratable acidity was 0.89 to 0.91%, and the pH was 4.55 to 4.65.

Then, mixed juice syrup, liquid fructose, 13 wt.%, Sugar 5 wt.%, Mixed juice concentrate 56Brix ^o 10.9% by weight of pectin, 1.0% by weight, fresh fruit mix Essence 0.1% and purified water 70% by weight at 30 ~ 35 ℃ Mixed with stirring, heat-treated with UHT (sterilized at 135 ° C for 2 seconds), and cooled.

Then, a mixture of 69.5% by weight of the above-mentioned lactic acid bacteria culture solution and 0.1% by weight of freeze-dried powder of a lyophilized powder or a 70% ethanol extract of a magnetic element hot water extract of Example 1 and 30.4% Homogenized and cooled to below 10 캜 to produce fermented milk.

<Example 4>

Production of Health Functional Foods Containing Self-extracting Ingredients as Active Ingredients

B vitamins B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid esters were added to 0.1% by weight of the lyophilized powder of the magnetic element hot-water extract of Example 1 or the lyophilized powder of the magnetic element 70% Nicotinic acid amide) and oligosaccharide were added in an amount of 10 parts by weight based on 100 parts by weight of the lyophilized powder obtained in Example 1, followed by mixing in a high-speed rotary mixer. 10 parts by weight of sterilized purified water was added to the mixture, mixed and molded into granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum drier at 40 to 50 DEG C and then passed through 12 to 14 mesh to prepare uniform granules. The granules thus prepared were extruded in suitable amounts to be purified or powdered or filled into hard capsules to prepare hard capsule products.

&Lt; Test Example 1 >

Cytotoxic measurement of osteoblast cell extracts

1-1. Cytotoxicity measurement of mouse skull osteoblast

end. Preparation of osteoblasts

Bone marrow cells were treated with 0.1% collagenase (Gibco), 0.1% dispase (Gibco), and 0.1% collagenase (PBS) in phosphate buffered saline (PBS) Were sequentially treated 6 times with an enzyme solution containing 3 to 6 times of cells in which osteoblast-specific cells were intensively collected and washed with serum free α-MEM. The washed cells were cultured in α-MEM containing 10% FBS for 2 days and then cultured for the first time. The collected cells were used for the experiment, and the recovered cells were diluted to a concentration of 1 × 10 ⁶ cells / &Lt; / RTI &gt;

I. Cytotoxicity measurement

To measure the cytotoxicity of the sample, the method of Green et al. (Green LM, Reade JL, Ware CF. Rapid colometric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lympocines. J. Immun. Meth. 70: 257 ), Was prepared 3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyltetrazolium bromide [3- -diphenyltetrazoliumbromide, MTT] analysis.

Specifically, the skull osteoblast of the cultured mouse was dispensed into a 96-well plate at a concentration of 1 × 10 ⁴ cells / 200 μl per well, cultured for 24 hours, and then the medium was removed.

Separately, the lyophilized powder of the hot-water extract of Example 1 or the lyophilized powder of the 70% ethanol extract of the magnetic element was added to 200 μl of the fresh MEM medium at a concentration of 3.12, 6.25, 12.5, 25, 50 and 100 μg / , And these were added to each well prepared above. After culturing for 48 hours in the culture medium to which the sample had been added, 10 μl of MTT (5 mg / ml) solution was added to each well and cultured for 4 hours. After the completion of the incubation, the supernatant was removed, 100 μl of DMSO was added to each well, and the resulting formazan crystals were dissolved. The absorbance was measured at 570 nm using a microplate reader. The cytotoxicity was expressed as a percentage of the absorbance of the test sample in the absorbance of the sample, and the results are shown in FIG. As the control (Con), those without the self-extract of the present invention were used.

1-2. Cytotoxicity of mouse osteoblast cell line MC3T3-E1 cell line

end. Preparation of cell line

MC3T3-E1 (mouse pre-osteoblast), which was used as a cell line, was purchased from ATCC (CRL-2594) and mixed with 10% fetal bovine serum (FBS) and 1X antibiotic (antibiotics, Anti-Anti, Gibco 15240) MEM (alpha-minimum essential medium, Gibco A10490) culture medium was a 5% carbon dioxide (CO ₂₎ incubator for one passage is present in two or three times a week in culturing using.

I. Cytotoxicity measurement

Except that MC3T3-E1 cell line, a skull osteoblast cell of mouse, was used instead of mouse osteoblast cell.

The results are shown in Fig.

1-3. Cytotoxicity measurement of human osteoblast cell line HOS cell line

end. Preparation of cell line

Hos cell (Human osteosarcoma cell) used as a cell line was purchased from Korean Cell Line Bank (KCLB-21543) and cultured with α-fetoprotein (FBS) supplemented with 10% fetal bovine serum and 1 × antibiotic MEM (alpha-minimum essential medium, Gibco A10490) culture medium was a 5% carbon dioxide (CO ₂₎ incubator for one passage is present in two or three times a week in culturing using.

I. Cytotoxicity measurement

Except that human osteoblast cell line HOS cell line was used instead of mouse osteoblast cell line.

The results are shown in Fig.

As can be seen from FIGS. 1 to 3, the hot-water extract of the magnetic element and the 70% ethanol extract of the magnetic element of the present invention were not cytotoxic even when treated at a high concentration.

&Lt; Test Example 2 &

Measurement of basic phosphatase activity of osteoblast cells in a rat

ALP (Alkaline phosphatase; ALP) activity test p - the amount of nitrophenol-nitrophenylphosphate ALP p is the hydrolysis product using that acts as a catalyst in the hydrolysis reaction of the (p -nitrophenyl phosphate) And the concentration of ALP is calculated by measurement.

The mouse skull osteoblasts of Test Example 1-1 were divided into well plates at a concentration of 5 × 10 ⁴ cells / well, and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. Then, the medium was removed .

Separately, the lyophilized powder of the hot-water extract of Example 1 or the lyophilized powder of the 70% ethanol extract of the magnetic element was added to the medium supplemented with 10% FBS and 1% antibiotic (penicillin-streptomycin) , And 12.5 占 퐂 / ml, and then dispensed into each well prepared above. After 7 days of culture, the culture medium was removed, and 0.1% Triton X-100 was added to dissolve the cells. 50 μl of the solution was taken out, and 100 μl of p -nitrophenyl phosphate (1.21 mM) was added thereto. The reaction was carried out at 37 캜 for 30 minutes, and 50 ㎕ of 0.2 N sodium hydroxide was added to stop the reaction.

Hydrolyzed from p-nitrophenyl phosphate - - p Through this process by measuring the amount of the nitrophenol was obtained the activity was calculated for the enzyme activity per unit protein content divided by the protein content. Absorbance was measured at 405 nm using a microplate reader (Bio-tek, Synergy HT, USA). Protein content was measured using a bovine serum albumin protein assay reagent. Enzyme activity was expressed as a percentage of the control. As the control, those without the self-extract of the present invention were used.

The results are shown in Fig. 4 and Fig.

As can be seen from FIGS. 4 and 5, the ALP activities of the self-extracts of the present invention were determined to be concentration-dependent and significantly increased in ALP activity, Showed about 70% or more increase in ALP activity. ALP is a biomarker expressed in the differentiation of osteoblasts. Thus, it can be seen that the self-extract of the present invention promotes differentiation of osteoblasts.

&Lt; Test Example 3 >

Measurement of bone calcification formation of osteoblast-derived cell extracts by Alizarin-red staining

Bone calcification ability is an important marker for osteoblast differentiation, and alizarin is stained in the matrix of mineralized cells, so that the amount of calcified and the degree of staining are proportional to each other.

The mouse skull osteoblast cells of Test Example 1-1 were divided into well plates at a concentration of 5 × 10 ⁴ cells / well and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. Then, the medium was removed.

For the induction of calcification, samples of the hot-water extracts of the magnetic element of Example 1 were added to the medium to which 50 μg / ml of ascorbic acid and 10 mM of β-glycerophosphate were added, 25, and 12.5 μg / ml, and the cells were dispensed into each of the prepared wells and cultured in an incubator until 14 days. After incubation, the medium was removed, washed with PBS and fixed with formalin.

The alizarin red (AR) solution was prepared by dissolving alizarin red (Sigma) in distilled water, adjusting the concentration to 40 mM and adjusting the pH to 4.2. After staining the cells, the cells were stained with AR solution (0.5 ml / well) for 10 minutes, washed 5 times with distilled water, and the unstained portions were washed with PBS and removed with a little wetness with PBS. The degree of formation was observed.

After confirming the formation of calcification, 0.5 ml of a 10 mM sodium phosphate (pH 7.0) solution to which 10% cetylpyridinium chloride was added was added to each well and the mixture was dissolved for 15 minutes. Absorbance was measured at 562 nm using a reader and the bone formation capacity was calculated based on the absorbance of the control group. As the control, those without the self-extract of the present invention were used.

The results are shown in Fig. 6 and Fig.

As can be seen from FIGS. 6 and 7, the effect of increasing the calcification of the osteoblasts was observed in a concentration-dependent manner. Therefore, it can be seen that the magnetic element extract of the present invention promotes differentiation of osteoblasts and has calcification-forming ability.

<Test Example 4>

Inhibition of osteoclast differentiation by cell extract

To examine the effect of the extract of the present invention on the growth and activity of osteoclast, osteoclast precursor cells were cultured and their expression and activity of TRAP (Tartrate resistance acid phosphatase), which is a labeling enzyme of osteoclast, were analyzed.

The TRAP enzyme is an enzyme with high activity when the osteoclast is osteoclastic and the enzyme secretion is increased and ATP, Nitrophenyl phosphate is present. The osteoclast cell cytochemical mark It is a marker enzyme.

On the other hand, osteoclasts are located in the lining of the bone and are the only polynuclear cells present in the bone tissue. RANKL forms osteoclast cells at the early stage of differentiation. However, when cells are fused and differentiated into polynuclear mature osteoclasts, And has a feature of forming a wrinkle film.

4-1. Inhibition of differentiation into mature osteoclasts using RANKL by hydrothermal extract

The osteoclast precursor cells were dispensed into 96 well plates (Well Plate) to give 1 x 10 ⁵ cells per well.

Separately, samples of the hot-water extract of the ruled element of Example 1 were added to α-MEM containing 30ng / ml Macrophage-colony stimulating factor (M-CSF, sigma) and 100ng / ml RANKL (Sigma) 100, 50, 25, 12.5, 6.25, and 3.12 占 퐂 / ml, and the wells were divided into the wells and cultured for 5 days. After cultivation, cells were fixed and TRAP staining was performed to examine osteoclast formation.

Specifically, 5 mg of substrate naphthol AS-MX phosphate (Sigma) and 25 mg of a pigment (Fast Red Violet LB salt) were dissolved in N, N-dimethylformamide (N , N-dimethylformamide) was added to 50 ml (pH 5.0) of 0.1N NaHCO ₃ buffer containing 50 mM Tartaric acid, and 70 μl of the TRAP staining solution was dispensed and observed under a microscope. As a control (Control), those without the self-extract of the present invention were used.

The results are shown in Fig.

4-2. Measurement of inhibition of mature osteoclast differentiation using RANKL by ethanol extract of 70% ethanol

The measurement was carried out in the same manner as in the measurement of inhibition of differentiation into mature osteoclasts using the RANKL of Example 4-1, except that a 70% ethanol extract was used.

The results are shown in Fig.

As can be seen from FIGS. 8 and 9, the inhibition effect of differentiation inhibition was confirmed when 50 or 100 μg / ml of hot-water extract of magnetic element was treated, compared with the control group treated with RANKL, which is a direct differentiation factor of osteoclast, The 70% ethanol extract showed 12.5, 25, 50, and 100 μg / ㎖ of inhibitory effect on the concentration-dependent differentiation.

4-3. RANKL-Induced Osteoclast Differentiation Suppressed by Massive Hydrothermal Extract

As a result of TRAP staining of Test Example 4-1, TRAP (+) polynuclear cells positive to TRAP (+) and having a polynuclear (more than 3 nuclei) And then counted by naked eyes. For accurate measurement, each well was divided into quarters and counted.

The results are shown in Fig.

4-4. RANKL-induced osteoclast differentiation inhibited by large-sized 70% ethanol extract

As a result of TRAP staining of Test Example 4-2, TRAP (+) polynuclear cells positive to TRAP (+) and having polynuclear (more than 3 nuclei) And then counted by naked eyes. For accurate measurement, each well was divided into quarters and counted.

The results are shown in Fig.

10 and 11, compared with the control group treated with RANKL, which is a direct differentiation factor of osteoclast, the case of treatment with hot water extract 50, 100 μg / ㎖, and the case of 70% ethanol extract 12.5, 25, 50, and 100 ㎍ / ㎖, respectively.

Claims (7)

Wherein the osteoblast cell has an activity of promoting osteoblast differentiation and osteoclast differentiation inhibitory activity.
The method according to claim 1,
Wherein the self-component extract is a hot-water extract, wherein the self-component extract has an activity of promoting osteoblast differentiation and inhibiting osteoclast differentiation.
The method according to claim 1,
Wherein the self-component extract is a 70% ethanol extract, which promotes differentiation of osteoblasts and inhibits osteoclast differentiation.
A pharmaceutical composition for preventing and / or treating bone diseases, which comprises the child element extract of any one of claims 1 to 3 as an active ingredient.
5. The method of claim 4,
The above-mentioned bone diseases include osteoporosis, Paget's bone disease, rickets, osteomalacia, renal osteodystrophy in patients with renal failure, rheumatic or degenerative bone disease, bone metastasis lesion, primary bone-derived tumors, periodontal disease, Inflammatory alveolar bone disease, and inflammatory bone resorption disease.
A food composition comprising the child element extract of any one of claims 1 to 3 as an active ingredient.
6. The method of claim 5,
Wherein the food composition is any one of fermented milk, functional beverage, and health functional food.

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