KR20040083631A - Ganoderma lucidum extract for the prevention and treatment of osteoporosis - Google Patents

Abstract

PURPOSE: Provided is Ganoderma lucidum extract for the prevention and treatment of osteoporosis. The Ganoderma lucidum extract contains a large quantity of lipid-soluble components and inhibits the induction of osteoporosis. Therefore, the Ganoderma lucidum extract is used as prophylaxis agents or therapeutics for osteoporosis. CONSTITUTION: Ganoderma lucidum extract is obtained by extracting Ganoderma lucidum with ethylalcohol at 70-80 deg.C for 3-5 hours, followed by concentration at 20-40 deg.C under reduced pressure. The Ganoderma lucidum extract contains lipid-soluble components and inhibits the induction of osteoporosis. The Ganoderma lucidum extract is added, as active ingredient, to prophylaxis agents or therapeutics for osteoporosis.

Description

Ganoderma lucidum extract for the prevention and treatment of osteoporosis}

The present invention relates to ganoderma lucidum ( Ganodema lucidum (Fr.) Karst.) Extract having an effect on the prevention and treatment of osteoporosis, and more specifically, to induce osteoporosis containing a large amount of fat-soluble components obtained by extracting ganoderma lucidum mushroom with ethyl alcohol It relates to a ganoderma lucidum extract with inhibitory activity.

Osteoporosis has been studied in the past mainly on bone minerals, namely, calcium and phosphorus metabolic abnormalities, and no progress has been made in identifying its pathogenesis. However, recently, the importance of research on metabolism of bone matrix proteins as well as minerals important for bone formation is emerging. For the treatment and prevention of osteoporosis, doctors usually recommend a diet containing calcium and postmenopausal women with estrogen or vitamin D to prevent osteoporosis or to slow the progression of osteoporosis. Calcium adjuvant, widely used as a treatment for osteoporosis, suppresses secretion of parathyroid hormone and prevents bone loss due to bone resorption, but it is known that individual differences in bone maintenance are severe (Heaney, RP, Principles of Bone biology , Academic Press, 1007). -1017, 1996). Hormone therapy with estrogen or calcitonin has been reported to increase bone density and reduce the incidence of rectal cancer, but side effects such as breast cancer, myocardial infarction and venous thrombosis have been reported (Nelson, HD et al ., JAMA , 288). : 872-881, 2002; Lemay, A., J. Obstet.Bynaecol.Can ., 24: 711-715, 2002). Bisphosphonates are attracting attention as new alternative therapeutic agents to inhibit osteoclasts, but lesions can be observed in the upper respiratory tract with incorrect oral administration (Fleisch, H., Principles of Bone biology , Academic Press, 1007-1017, 1996 Cryer, R. and Bauer, DC, Mayo. Clin. Proc ., 77: 1031-1043, 2002)

Therefore, there is an urgent need for the development of new substances effective in the prevention and treatment of osteoporosis due to the new action and skeletal structure and less toxicity and side effects. In addition, since these new substances are very likely to be found in natural products that have been applied to folk medicine since ancient times, attempts are being actively made to produce new drugs from natural products, particularly herbal medicines.

Osteoporosis is not a symptom itself but rather various fractures that are easily caused by bone weakness, especially femoral fractures or vertebral fractures, which limit long-term activities and lead to a healthy life. It is a problem because.

On the other hand, bone tissue is a dynamic tissue in which the formation by osteoblasts and the destruction and absorption by osteoclasts are constantly repeated. There are many uncertainties about the mechanism of bone absorption and formation, but osteoporosis is a disease caused by excessive absorption of bone than bone formation due to a breakdown of the balance between bone absorption and bone formation. Therefore, the development of bone resorption inhibitors is currently active.

Ganoderma lucidum (Fr.) Karst. Is a perennial mushroom belonging to the Polyporaceae family.It is a supernatant to the herbaceous and new agricultural herbaceous plants of Lee Si-jin, China's best herb, more than 2,000 years ago. It is described as effective for the treatment of action, arthritis, remission, bronchitis and the like. In oriental medicine, it has been used in combination with other herbal medicines such as arteriosclerosis, hypertension, stroke, angina, various cancers, myasthenia gravis, acute bronchitis, etc. because of its pharmacological effects such as nourishing tonic, detoxification convergence, droplets, and blood lipid reduction. Recently, the pharmacological effects of Ganoderma lucidum have been reported to be excellent pharmacological effects related to anticancer, neurological, circulatory, immunomodulatory and anti-HIV effects. The main ingredient is lanostane, a belonging to the triterpenoids, a polysaccharide of 1,050 kDa and a bitter taste unique to Ganoderma lucidum. Lucidenic acid D> ganoderic acid A> Gummy was found in the order of lucidone A> lucideic acid A> ganoderic acid C> lucidone C. These mushrooms are known to exhibit various physiological activities, including hepatotoxicity of ganoderic acid R and S, histamine release of ganoderic acid C and D, cholesterol lowering effect of ganoderic acid B and TO, and ganoderic acid B. , Antihypertensive action of D, F, H, K, S and Y are known.

Accordingly, the inventors found that ganoderma lucidum extract by hydrothermal or ethyl alcohol extraction method had osteoporosis-induced inhibitory activity while conducting various physiological activities of ganoderma lucidum mushroom, and ganoderma lucidum by bioassay-directed fractionation. The fat-soluble component of the mushroom extract confirmed the potent osteoporosis-induced action, and completed the present invention by revealing that the Ganoderma lucidum extract can be usefully used as a therapeutic or preventive agent and a health food for osteoporosis.

An object of the present invention is to provide a Ganoderma lucidum extract containing a fat-soluble component having an osteoporosis-inducing inhibitory activity, and a pharmaceutical composition or health food for osteoporosis prevention or treatment containing the Ganoderma lucidum extract as an active ingredient.

1 is a graph showing the effect on osteoblast proliferation according to the concentration of Ganoderma lucidum extract,

K6EB: Normal butanol fraction of Ganoderma lucidum ethanol extract,

K6EEa: ethyl acetate fraction of Ganoderma lucidum ethanol extract,

Figure 2 is a graph showing the effect of Ganoderma lucidum mushroom extract on collagen, basic phosphatase, osteopontin and osteocalcin RNA expression of RCC cells,

COL: collagen, ALP: basic phosphatase,

OPN: Osteopontin OC: Osteocalcin,

K6EB-0.5: 0.5 µg / ml normal butanol fraction of Ganoderma lucidum ethanol extract,

K6EB-5: 5 ㎍ / ml normal butanol fraction of Ganoderma lucidum ethanol extract,

K6EEa-0.1: 0.1 µg / ml ethyl acetate fraction of Ganoderma lucidum ethanol extract,

K6EEa-1: 1 μg / ml ethyl acetate fraction of Ganoderma lucidum ethanol extract,

Figure 3 is an electrophoresis picture showing the effect of Ganoderma lucidum extract on OPG expression of osteoblasts (concentration unit is ㎍ / ㎖),

K6EB: Normal Butanol Fraction from Ganoderma Lucidum Ethanol Extract

K6EEa: Ethyl Acetate Fraction from Ethanol Extract of Ganoderma lucidum Mushroom

Figure 4 is a graph showing the effect of Ganoderma lucidum extract on the formation of osteoclasts (concentration unit is ㎍ / ㎖, MNCs means multinucleated cells),

5 is a graph showing the effect of Ganoderma lucidum mushroom extract on osteoclast activity (concentration unit is ㎍ / ㎖, MNCs means multinucleated cells),

Figure 6 is a graph showing the expression of the transcription factor (Runx2) for differentiation compared to the control when the Ganoderma lucidum extract was treated with C2C12 cells inserted with the 6xOSE2-Luc vector (concentration unit is ㎍ / ㎖),

In order to achieve the above object, the present invention provides a ganoderma lucidum extract having an osteoporosis-inducing activity containing a large amount of fat-soluble components by extracting and concentrating ganoderma lucidum with ethyl alcohol.

The present invention also provides a pharmaceutical composition or health food for treating or preventing osteoporosis containing the Ganoderma lucidum extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides ganoderma lucidum extract having inhibitory activity against osteoporosis-induced fat-containing components by extracting and concentrating ganoderma lucidum with ethyl alcohol.

At this time, the ethyl alcohol extraction is preferably made for 3 to 5 hours at 70 to 80 ℃, the concentration is preferably made of vacuum decompression at 20 to 40 ℃.

In order to further isolate the active ingredient of the extract, although not particularly limited to the separation method that can be used, such as organic solvent extraction, chromatography, it is preferable to extract with ethylacetate (ethylacetate), as in the embodiment of the present invention, More preferred is extraction with n-butanol. In addition, the extraction with ethyl alcohol is preferably repeated three or more times.

The ganoderma lucidum extract is characterized by containing a large amount of fat-soluble components. Ganoderma lucidum extract having osteoporosis-induced inhibitory activity of the present invention extracts ganoderma lucidum mushroom with ethyl alcohol, and then additionally extracts the extract with an organic solvent to obtain a fraction containing a large amount of fat-soluble components, which is concentrated under reduced pressure to obtain final ganoderma lucidum. Prepare mushroom extracts.

In one embodiment of the present invention, in order to investigate the therapeutic effect of osteoporosis, such as Ganoderma lucidum extract or a compound comprising the same, tests were performed on primary osteoblasts of human osteosarcoma cells and rat osteoblast group (RCC). As a result, in the proliferation of osteoblasts, the expression level of mRNA for the characteristic genes of osteoblasts, the expression level of OPG (osteoprotegerin) which inhibits the production of osteoclasts by osteoblasts, and the expression of transcription factors indicating osteoblast differentiation, Ganoderma lucidum extract seems to improve the function of osteoblasts (see FIGS. 1 , 2 , 3 and 6 ). In addition, the Ganoderma lucidum extract significantly inhibits osteoclast function by greatly reducing the formation of TRAP (+) multinucleated cells and the activity of osteoclasts, which are considered osteoclasts, and contribute more to the reduction of bone tissue destruction ( FIGS. 4 and 5). Reference). Therefore, Ganoderma lucidum extract of the present invention promotes the proliferation and differentiation of osteoblasts, reduces the formation and activity of osteoclasts and is effective in the treatment and prevention of osteoporosis. In particular, as described in the Examples of the present invention, it is determined that the Ganoderma lucidum mushroom extract contributes more to blocking the development and progression of osteoporosis by inhibiting the formation and activity of osteoclasts.

The present invention also provides a pharmaceutical composition for preventing or treating osteoporosis containing the Ganoderma lucidum extract as an active ingredient.

The pharmaceutical composition comprising Ganoderma lucidum extract of the present invention may be administered orally or parenterally during clinical administration and may be used in the form of a general pharmaceutical formulation.

That is, the composition of the present invention can be administered in various oral and parenteral dosage forms in actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used Is prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose ( Sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used. In addition, calcium or vitamin D ₃ may be added to improve the efficacy as a prophylactic or therapeutic agent for osteoporosis.

Dosage units may contain, for example, one, two, three or four times, or 1/2, 1/3 or 1/4 times the individual dosage. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose.

The effective dose of the composition is 33.3-83.3 mg / kg, preferably 33.3-50 mg / kg, and may be administered 2-3 times a day. The composition as described above is not necessarily limited to this, and may vary depending on the condition of the patient and the degree of development of neurological diseases.

As a result of conducting oral toxicity and mouse intraperitoneal administration of the pharmaceutical composition comprising Ganoderma lucidum extract of the present invention, 50% lethal dose (LD ₅₀ ) by oral toxicity test is at least 5,000 mg / kg or more It turned out to be a safe substance.

The present invention also provides a health food containing the Ganoderma lucidum extract.

When the Ganoderma lucidum extract of the present invention is used as a food additive, the Ganoderma lucidum extract may be added as it is, or may be used together with other food or food ingredients, and may be appropriately used according to a conventional method. The blending amount of the active ingredient can be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment). In general, in the preparation of food or beverage, the Ganoderma lucidum extract of the present invention is added in an amount of 0.1 to 15% by weight, preferably 0.2 to 10% by weight based on the raw materials. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. Is sure.

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the present invention is not limited to the contents of the present invention.

Example 1 Preparation of Ganoderma lucidum Extract

Ganoderma lucidum mushroom was cultivated in Korea and was purchased from a commercial building. The sliced ganoderma lucidum was ground into powder, 200 g of powder was placed in a 3 L flask, and heat extraction was repeated three times for 4 hours while stirring at reflux at 78.5 ° C. using 2,000 ml of ethanol. After filtering the heated extract and vacuum concentrated under reduced pressure using a vacuum rotary evaporator at 40 ℃ or less to obtain the first crude extract containing 11.4 g of Ganoderma lucidum powder (yield for Ganoderma lucidum powder: 5.7%). Ganoderma lucidum extract containing fat-soluble components was isolated by the above extraction method. The extract was dissolved in 20 ml of ethanol and suspended in 500 ml of distilled water, and then the solvent was fractionated three times using a separatory funnel with 500 ml of ethylacetate at room temperature, and named 'K6EEa'. . The K6EEa was additionally fractionated three times by using a fractionation funnel with 500 ml of normal butanol (n-butanol) at room temperature and named 'K6EB'. In the following examples, the extracts fractionated with K6EEa and K6EB were concentrated and lyophilized, and then diluted to 5 g / L in ethanol, and the following osteoblast and osteoclast experiments were performed in vitro . .

<Example 2> Osteoblast proliferation experiment by Ganoderma lucidum extract fraction

In order to determine the effect of Ganoderma lucidum extract fraction isolated in Example 1 on the proliferation of osteoblasts and the increase of cell activity, the following experiments were carried out.

<2-1> Osteoblast Selection and Cell Culture

The following three cells were used to evaluate the effect of Ganoderma lucidum mushroom extract on osteoblast proliferation. Human cells include MG-63 cells (ATCC No. CRL-1427), a cell line derived from human osteosarcoma, and mouse muscle cells, C2C12 cells (ATCC No. CRL-1772), ATCC (Rockville, USA). ) Was used in the experiment after incubation in a medium in which 10% FBS (fetal bovine serum) was added to DMEM (Gibco, BRL, USA).

In addition, as the primary cultured cells, osteoblast group (RCC) obtained from the cranial canal of Sprague-Dawley rat was used. That is, on the 19th day of birth, the frontal and parietal bones were extracted from the cranial canal of the Sprague-Dawley rats, and then each of the enzyme solution containing 0.1% collagenase, 0.05% trypsin, and 0.5 mM EDTA, respectively, at 37 ° C. The cells of groups I to V were separated by continuous treatment for minutes, 10 minutes, 10 minutes, 20 minutes, and 20 minutes, and cells of groups IV and V, in which cells with the characteristics of double osteoblasts, were predominantly mixed, 10 After primary culture in DMEM containing% FBS cells were collected with trypsin-EDTA solution and the number of cells was used for each experiment.

<2-2> Measurement of Osteoblast Proliferation

In order to examine the effect of Ganoderma lucidum extract on the proliferation of osteoblast cells, using the three osteogenic prepared by a method described in the above Example <2-1> introduced into DNA during the cell proliferation, cell ³ H- The amount of thymidine was measured. Specifically, osteoblasts were dispensed to 2 x 10 ⁴ cells per well of a 24-well plate, and replaced with DMEM containing 1% FBS the next day. Ganoderma lucidum extract prepared by diluting continuously in concentrations of 10 ⁻⁴ , 10 ⁻³ , 10 ⁻² , 10 ⁻¹ , 1 or 10 μl / ml in the replaced culture medium was added to each concentration and incubated for 48 hours. After incubation with 3 μCi / well of ³ H-thymidine for the last 4 hours, the cells were washed with phosphate buffered saline (PBS) and treated with ice-cold 5% trichloroacetic acid (TCA) insoluble fraction. Was separated and dissolved in 0.1 M NaOH. ³ H-thymidine influx was measured by taking a double fraction and measuring radioactivity on a liquid scintillation counter.

The degree of osteoblast proliferation according to various Ganoderma lucidum extract concentrations by the above process is shown in FIG. 1 . When treated with low concentration of Ganoderma lucidum extract, there was no significant difference from the control without Ganoderma lucidum extract, but osteoblast cells in K6EEa or control were proportional to the concentration of 10 ㎍ / ml to 50 ㎍ / ml in K6EB. It was confirmed that the proliferation was significantly higher ( FIG. 1 ).

<2-3> mRNA expression for characteristic genes of osteoblasts (type 1 collagen, basic phosphatase, osteopontin and osteocalcin)

In order to evaluate the effect of Ganoderma lucidum extract on osteoblast differentiation, after preparing and incubating the cranial canal osteoblast cells of Sprague-Dawley rat by the method described in Example <2-1>, Subcultured cells were collected and aliquoted into 60 mm tissue culture dishes at a density of 63 × 10 ⁴ / dish. Osteoblasts were cultured in α-minimum essential medium containing 10% FBS, and 50 ㎍ / ml of ascorbic acid and β-glycerophosphate from the third day of culture. 10 mM was added to induce differentiation of osteoblasts. From the day after the start of culture, K6EB was added to the culture dish of osteoblasts at concentrations of 0.5 and 5 μg / ml and K6EEa at concentrations of 0.1 and 1 μg / ml, and the same concentration was changed every two days. Ganoderma lucidum extract fractions were added and cultured for 12-15 days when calcified nodules were produced in osteoblasts. To examine the effect of Ganoderma lucidum extract, total RNA was isolated using the Easy Blue ^TM total RNA isolation kit after 3 days, 7 days of culture and 14 days of culture. . Of these, 25 μg of total RNA was subjected to electrophoresis on a 1.2% agarose / formaldehyde denaturing gel and transferred to a nylon filter (Hybond-N). Thereafter, a prehybridization reaction was performed in an ExpressHyb ^™ hybridization solution. Pro-α1 (I) -collagen, basic phosphatase, osteopontin, or osteocalcin probes labeled with ³² P-dCTP were then isolated using a megaprime DNA labeling kit. Hybridization in the contained hybridization solution for 1 hour, washed twice with wash solution 1 (2 X SSC, 0.05% SDS), once in washing solution 2 (0.1 X SSC, 0.1% SDS) and then Autoradiography was performed and the comparison of the expression level of the protein was analyzed using a Fuji Bas 1500 image analyzer.

As a result of measuring the type 1 collagen, basic phosphatase, osteopontin, and osteocalcin mRNA expression of osteoblasts examined by the above process, Ganoderma lucidum extract promoted the expression of type 1 collagen, basic phosphatase, and osteopontin. It was confirmed that ( Fig. 2 ). At this time, the basic phosphatase is an enzyme bound to the cell membrane of osteoblasts and is used as a marker of osteoblasts and is thought to be involved in various stages of bone tissue formation (Farley, JR and Baylink, D., Metabolism , 35: 563-571, 1986). Collagen type 1, basic phosphatase, osteopontin and osteocalcin are known as characteristic genes for osteoblasts (Gerstenfeld, LC et al ., Develop. Biol ., 122: 49-60, 1987; Yoon, K. et al ., Biochem. Biophys. Res. Commun. , 148: 1129-1136, 1987).

<2-4> Expression of OPG (osteoprotegerin) that inhibits osteoclast production by osteoblasts

The MG63 cell line was cultured confluent in a 60 mm tissue culture dish, followed by adding 2 ml of serum-free DMEM containing Ganoderma lucidum extract at concentrations of 1, 4, 20 and 100 ㎍ / ml for 16 hours. After incubation, the supernatant was harvested. The culture supernatant was concentrated with 20-25% trichloroacetic acid (TCA), dissolved in SDS-PAGE sample buffer, and electrophoresed on 10% acrylamide SDS-PAGE gel. Samples were transferred from gels to PVDF membranes by standing at 250 kPa for 1 hour after electrophoresis. After the sample was transferred to the blocking buffer (5% skim milk) for 1 hour, the anti-OPG antibody (R & D system), which is a primary antibody, was diluted 1:25 in a blocking buffer and added to a solution prepared at room temperature. It was left for about 2 hours. After washing the membrane twice with TBST solution (100 mM Tris-Cl, 0.9% NaCl, 0.1% tween 20, pH 7.5) for 15 minutes, the secondary antibody (also known as Promega) with alkaline phosphate was added. The membrane was left for 1 hour in a solution diluted in blocking buffer at 7000. After washing the membrane three times for 20 minutes with the TBST solution, the substrate was reacted for 15 minutes and then analyzed using RAS (Fujifilm).

As a result of measuring the expression of OPG, a substance that inhibits the production of osteoclasts in osteoblasts, the concentration of osteoblasts was increased in proportion to the concentration of up to 20 ㎍ / ml as the concentration of K6EB and K6EEa extracts of Ganoderma lucidum were increased. There was a tendency to promote the expression of OPG, and at higher concentrations the expression efficiency of OPG appeared to decrease relatively ( FIG. 3 ).

<2-5> Expression of major transcription factor (Runx2) in osteoblast differentiation

Transient transfection of C2C12 cells (ATCCNo. CRL-1772) with a 6xOSE2-Luc vector inserted into the pGL3 promoter vector, a series of six OSE2 DNA sequences bound by Runx2, an essential transcription factor for bone formation The method of determining the expression level of Runx2 as luciferase activity by administering the test subject fraction was selected.

In order to promote bone formation, osteoblast differentiation must be preceded. In particular, in order to promote osteoblast differentiation, expression of Runx2, which is a transcription factor for differentiation, is essential. This increases the expression of several genes (osteocalcin, osteopontin, type 1 collagen, bone sialoprotein) that osteoblasts require. The promoter of these genes contains the response element of Runx2 (also called osteoblast specific factor binding element), and its central consensus sequence is PuACCPuCA (where P is the purine base). And u represents a pyrimidine base). This increased osteoblastic gene expression in osteoblasts has been shown to be involved in the promotion of bone formation (Lee, KS., Et al ., Mol. Cell. Biol ., 2000 Dec; 20 (23): 8783-92 Park, MH, et al ., J. Bone Miner Res ., 2001 May; 16 (5): 885-92).

The detailed experimental method is as follows.

Day 0

Each C2C12 cell is seeded at 1 × 10 ⁵ density per well of a 6 well plate and then incubated in a 5% CO ₂ incubator for 24 hours.

Day 1

(1) p6xOSE2-Luc: lipopeptamine complexes were prepared using Lipofectamine. Specifically, Solution I was prepared by mixing 0.5 μg of p6xOSE2-Luc, 3 μl of PLUS reagent, and 100 μl of serum-free DMEM medium per 6 well plate at room temperature for 15 minutes.

Next, Solution II was prepared, including DMEM without 3 μl of lipofectamine and 100 μl of serum, and then the solution I and solution II were mixed and reacted at room temperature for 15 minutes to allow p6xOSE2-Luc: lipo. Pectamine complexes were prepared.

(2) transformation

1) After removing the culture medium of the cells for 15 minutes, the cells were washed with DMEM without serum. DMEM without 800 μL of serum was added per well of a 6 well plate.

2) 207 μl of p6xOSE2-Luc: lipopeptamine complex was added to the wells, followed by incubation in a 5% CO ₂ incubator for 3 hours.

3) After 3 hours of incubation, 1 ml of DMEM containing 30% FBS was added per well, followed by incubation in a 5% CO ₂ incubator for 24 hours.

Day 2

After removing the culture medium of the cells, the cells were washed with DMEM without serum. Treatment with serum-free DMEM with 2 ml of bFGF added at 10.0 ng / ml per well of 6-well plate, control group treated with serum-free DMEM without bFGF, followed by 5 hours of treatment for 24 hours. Cultured in a% CO ₂ incubator.

Day 3

After 24 hours of incubation with PBS solution, cells were treated with 500 μl of bright glo lysis buffer (Promega, WI, USA) and then bright glo substrate (Promega, WI, USA). After reacting for 5 minutes by adding the same amount, Luciferase activity was measured using a luminometer (Junior, EG & G BERTHOLD, Australia).

As a result of measuring the effect of Ganoderma lucidum extract on p6xOSE2-Luc-temporally transduced C2C12 cells, it was confirmed that the expression of Runx2 gene was significantly increased compared to the control group ( FIG. 6 ).

<Example 3> inhibition of osteoclast proliferation by Ganoderma lucidum extract

In order to confirm the inhibition of the production of osteoclasts by the Ganoderma lucidum extract and reduction of cell activity, the present inventors performed the following experiments.

<3-1> Pure separation of osteoclast progenitor cells and differentiation into mature osteoclasts and treatment of Ganoderma lucidum extract

In order to separate mouse bone marrow cells, 7-9 week old male mice were sacrificed with cervical torsion, followed by sterile extraction of the femur and tibia, removal of soft tissues, and cutting of both ends of the long bone, followed by a 26G needle. Inject 1 ml of enzyme solution containing 0.1% collagenase (Gibco), 0.05% trypsin, and 0.5 mM EDTA (Gibco) into the bone marrow cavity, remove the bone marrow, stir for 30 minutes, collect the bone marrow cells, and collect 10% FBS. After incubation for 24 hours in α-Minimum Medium (α-MEM) containing the unattached cells were obtained. Unattached cells, which are osteoclast progenitor cells, were cultured by aliquoting 2 × 10 ⁵ cells per well. Ganoderma lucidum extract prepared in Example 1 in α-MEM containing 20 ng / ml macrophage-colony stimulating factor (Peprotech, USA) and 30 ng / ml RANKL (Peprotech, USA) during 8 days of incubation Treated and incubated. After completion of the culture, in order to examine the production of osteoclasts, the cells were fixed and subjected to TRAP staining. In addition, in order to examine the activity of osteoclasts, the cells were detached and observed at the site of absorption.

<3-2> Inhibition of the production of multinucleated cells that are TRAP (+)

After cell culture, adherent cells were washed with PBS and then fixed with citrate-acetate-formaldehyde for 5 minutes, naphthol AS-BI phosphate, fast garnet GBC. TRAP staining was performed by incubating the solution and 37 mM acetate buffer (pH 5.0) containing 7 mM tartrate buffer (pH 5) for 1 hour. At this time, TRAP (+) multinucleated cells having three or more nuclei were considered as osteoclasts.

Osteoclasts are derived from cells of the monocyte / macrophage lineage in the bone marrow, which are circulated into the blood and are known to proliferate and proliferate in the endothelial layer to form multinucleated cells (Scheven, BAA et al. , Nature , 321: 79-81, 1986), osteoclasts have a tartrate-resistant acid phosphatase (TRAP), an acid phosphatase that is characteristically resistant to tartrate, which is different from other bone tissue cells. It is known to be used as a distinguishable cytochemical marker of osteoclasts (Minkin, C., Calcif. Tissue Int. , 34: 285-290, 1982).

In the present invention, bone marrow known to have osteoclast progenitor cells was used to induce differentiation of osteoclasts, and the cells were considered as osteoclasts in positive (+) and multinuclear cells in TRAP, while culturing osteoclast precursor cells. The Ganoderma lucidum extract prepared in 1 was treated and cultured for 8 days, and then the change in the number of TRAP-positive multinucleated cells was measured. As a result of measuring TRAP activity, which is a marker enzyme of osteoclasts, it was confirmed that the addition of Ganoderma lucidum extract significantly reduced the number of TRAP (+) multinucleated cells from 100% to less than 10% compared to the control group. From the above results, it was confirmed that the Ganoderma lucidum extract of the present invention has an inhibitory effect on the production of osteoclasts ( FIG. 4 ).

<3-3> Inhibition of osteoclast activity

In order to confirm the growth and activity of osteoclasts, osteoclast progenitor cells were cultured on a plate coated with hydroxide apatite (OAAS, OCT Inc.), and the TRAP activity and resorption pit, markers of osteoclasts, were observed. . Specifically, the culture medium was removed after cell culture. After culturing the cells, the OAAS plate was washed with distilled water in order to remove the adhered cells. Then, 5% sodium hypochlorite solution was added to the solution and left for 5 minutes. Observations were made using Image pro plus. In other words, to examine the activity of osteoclasts that are mainly responsible for bone resorption in bone tissue, Ganoderma lucidum extract was extracted by culturing progenitor cells of osteoclasts using calcium and phosphate-coated plates made similar to the mineral part of bone tissue. Upon treatment, it was observed whether there was a change in absorption area.

As a result, when the Ganoderma lucidum extract was added, it was confirmed that the osteoclast progenitor cells were cultured on a plate coated with calcium-phosphate to significantly reduce the activity and absorption of multinucleated cells showing TRAP (+) ( FIG. 5 ).

Example 4 Acute Toxicity Test by Ganoderma Lucidum Extract

The Ganoderma lucidum used in the present invention was widely used for food, and thus it was determined that there was no problem in stability.

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended orally at a dose of 5 g / kg in each of K6EFa and K6EB of the present invention suspended in 0.5% methylcellulose solution. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological examinations were performed.

As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, all Ganoderma lucidum extract did not show toxic changes up to 5 g / kg in rats, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of more than 5 g / kg.

As described above, Ganoderma lucidum extract according to the present invention has excellent osteoblast production and osteoclast suppression ability, Ganoderma lucidum extract can be used as a health food or not only as a therapeutic or preventive agent for osteoporosis.

Claims (8)

Ganoderma lucidum mushroom extract having an osteoporosis-inducing activity containing fat-soluble components by extracting Ganoderma lucidum with ethyl alcohol. The extract of claim 1, wherein the ethyl alcohol extraction is performed at 70 to 80 ° C. for 3 to 5 hours. The extract according to claim 1 or 2, wherein the concentration is performed under reduced pressure at 20 to 40 ° C. The extract of claim 1, wherein the extract is additionally extracted with ethyl acetate. The extract of claim 4, wherein the extract is additionally extracted with normal butanol. The extract according to claim 1, wherein the extracting with ethyl alcohol is repeated three or more times. A pharmaceutical composition for treating or preventing osteoporosis, comprising the extract of any one of claims 1 to 6 as an active ingredient. Health food containing the extract of any one of Claims 1-6.