KR20160057525A

KR20160057525A - composition for the prevention and treatment ofosteoporosis containing Acyranthes bidentata Blume and ginseng extract

Info

Publication number: KR20160057525A
Application number: KR1020140157944A
Authority: KR
Inventors: 이상원; 김영옥; 김진성
Original assignee: 대한민국(농촌진흥청장)
Priority date: 2014-11-13
Filing date: 2014-11-13
Publication date: 2016-05-24

Abstract

The present invention relates to a composition for alleviating and preventing osteoporosis, comprising an Acyranthes bientada Blume extract and a ginseng extract as an active ingredient, which exhibits combined effects of effects in inhibiting the differentiation of an osteoclast, effects in increasing bone density, and effects in densifying a microstructure of the bone. The composition can prevent the cytotoxicity of a high-concentration ginseng extract in the case of mixing and using the same, in comparison with the case of separately using each extract in the present invention. In addition, the composition can be broadly used as a health food product and a medical product, useful for preventing or treating osteoporosis.

Description

[0001] The present invention relates to a composition for prevention and treatment of osteoporosis, which comprises as an active ingredient an extract of Aspergillus oryzae and a ginseng extract,

The present invention relates to a pharmaceutical composition for prevention and treatment of osteoporosis and a health functional food composition for preventing or ameliorating osteoporosis, which comprises an extract of rubella ginseng and an extract of ginseng as an active ingredient. The present invention relates to an effect of inhibiting osteoclast activity, The present invention relates to a method for inhibiting cytotoxicity of a high concentration ginseng extract by combining the extracts of the present invention with a compound of the present invention, And can be widely used as medicines and health foods useful for prevention or treatment of osteoporosis.

A bone is a calcified connective tissue associated with muscles. The surface is a thick, hard calcified tissue that plays a role in balancing the body and protects organs. The inside of bone is bone marrow tissue, which is the center of calcium metabolism. The bone consists of an organic substance such as collagen, osteocalcin, osteonectin, and inorganic substance such as calcium, phosphorus, and fluorine, and moisture. In the living body, bone formation and bone resorption are always occurring. Bone formation is promoted by small amounts of parathyroid hormone, androgens, estrogens, fluorine, phosphorus, and bone resorption is promoted by physical decompression, weightlessness, a large amount of parathyroid hormone, corticosteroids, Therefore, balance between them is important in bone metabolism.

Osteoporosis is a disease caused by the collapse of the balance between bone resorption and bone formation, which is caused by excessive bone resorption over osteogenesis. Osteoporosis is a disease caused by reduced lime of bone tissue and thinness of bone, As the bone marrow cavity is widened and the symptoms progress, the bone is weakened, so it is likely to fracture even in small impacts. Bone tissue is a dynamic tissue that is formed by osteoblasts and is constantly repetitively absorbed and destroyed by osteoclasts.

The homeostasis of bone is maintained by continuously controlling the bone remodeling by the equivalent action of bone resorption by osteoclast and osteoblast formation (bone formation) .

However, excessive activity of osteoclasts or a decrease in osteoblast activity leads to an imbalance in the remodeling process, leading to adult skeletal diseases such as osteoporosis. In order to alleviate this imbalance, methods for suppressing excessive activity of osteoclasts, promoting osteoclast activity, inhibiting osteoclast activity and promoting osteoclast activity are generally used, and they are used for treatment of osteoporosis As well as for the treatment of the disease.

Osteoclasts are cells derived from mononuclear cells of hematopoietic stem cells. Mouse RAW264.7 mononuclear cells are differentiated into multinucleated osteoclasts by fusing with RANKL (receptor activator of nuclear factor κB (RANK) ligand) : Boyle WJ, Simonet WS, Lacey DL, Osteoclast differentiation and activation, Nature, 2003 May 15; 423 (6937): 337-42). This differentiation process promotes the activation of mitogen-activated protein kinase (MAPK) by RANKL binding to RANK in the extracellular region, and this is because the transcription factor NF-κB enters into the nucleus and binds to osteoclast differentiation-related TRAP tartrate-resistant acid phosphatase,

MMP-9 (matrix metalloproteinase-9), and c-Src tyrosine kinase. The polynuclear osteoclast formed by this process can absorb mineralized bone. Furthermore, binding of RANKL to RANK promotes the activity of TRAF6 (tumor necrosis factor receptor-associated factor 6)

MAPK, or NF-κB, AP-1, and NFATc1 (Lee ZH, Kim HH, Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts. Biochem Biophys Res Commun. 2003 May 30; 305 (2): 211-4). In particular, it has been reported that AP-1 or NFAT, which is specific for osteoclast differentiation, can be regulated by ERK and NF-κB which are found to be signal transduction molecules that play an important role in osteoclast survival and bone resorption capacity [Takayanagi H, Induction and activation of the transcription factor NFATc1 (NFAT2), which is an inducible factor of NFAT2 (NFAT2), is involved in the regulation of NFAT1 (NFAT2) integrase RANKL signaling in terminal differentiation of osteoclasts. Dev Cell. 2002 Dec; 3 (6): 889-901; Asagiri M, Sato K, Usami T, Ochi S, Nishina H, Yoshida H, Morita I, Wagner EF, Mak TW, Serfling E, Takayanagi H. Autoamplification of NFATc1 expression determines its essential role in bone homeostasis. J Exp Med. 2005 Nov 7; 202 (9): 1261-9). Thus, blockade of the signaling pathway activated by RANKL has been recognized as one of the therapeutic approaches for the treatment of bone diseases including osteoporosis.

Osteoblast cells are formed from mesenchymal stem cells. Mineralization such as calcium formation by osteoblast differentiation not only maintains bone strength but also plays a very important role in the homeostasis of calcium and hormone metabolism throughout the body. Calcium formation by osteoblast differentiation is regulated by vitamin D and parathyroid hormone. Bone morphogenesis by osteoblast differentiation induces bone morphogenetic protein (BMP), Wnt, MAP The interaction of various signal transduction systems such as kinase, calcineurin-calmodulin kinase, NF-κB and AP-1 has been shown to be involved in the differentiation of osteoblastic cells with alkaline phosphatase ; ALP) is synthesized in the early differentiation stage and then osteopontin, osteocalcin, type I collagen, etc. associated with mineralization are synthesized. Since the promoter for activating such osteoblast cells generally requires long-term administration to the patient, it is preferable that the osteoblast cell is less toxic and can be administered orally. Development of osteoblast-activating drugs and functional foods without toxicity is required.

Osteoporosis is a cause of 15% of the deaths of the elderly as a result of various fractures, especially femoral fractures or vertebral fractures, which are easily caused by the weakening of the bone rather than the symptom itself, . The bone mass is affected by various factors such as genetic factors, nutritional intake, hormonal changes, exercise and lifestyle differences, and the causes of osteoporosis include age, lack of exercise, low birth weight, smoking, low calcium diet, Ovariectomy, and the like are known.

Although there are individual differences, blacks have a lower bone resorption level than blacks, and bone mass is higher. Usually, bone mass is highest at 14-18 years old and decreases about 1% per year at old age. Especially in women, bone reduction continues after 30 years of age, and bone turnover is rapidly progressed by hormonal changes in menopause.

Thus, osteoporosis is an unavoidable symptom for elderly people, especially postmenopausal women. In the developed countries, as population ages, interest in osteoporosis and its therapeutic agents is gradually increasing.

It is also known that there is a market of about $ 130 billion related to the treatment of bone diseases worldwide and it is expected to increase further in the future. Therefore, each research institute and pharmaceutical company in the world invests heavily in the development of bone disease drugs Currently, the development of bone resorption inhibitors is actively underway.

In the past, osteoporosis was mainly related to the bone mineral, calcium and phosphorus metabolism. However, no major progress has been made in the identification of the mechanisms underlying the onset of osteoporosis.

Currently, there are bisphosphonates (alendronate, etidronate), hormone preparations (raloxifene), vitamin D preparations, calcitonin preparations, and calcium preparations. However, bisphosphonate preparations have poor water uptake and are difficult to take and cause esophagitis, and hormone preparations should be used for a lifetime. When administered for a long time, adverse effects such as breast cancer, uterine cancer, gallstone and thrombosis appear. Vitamin D preparations are expensive and effective Calcitonin preparations are expensive, difficult to administer, and calcium preparations have few side effects, but they are limited to preventive effects rather than treatment.

Osteoporosis can not be treated by only short-term administration of the drug, and long-term administration of the drug is essential. Therefore, there is a need for a new substance having excellent drug efficacy without the above-mentioned side effects when the drug is administered for a long period of time.

Recently, in order to minimize such side effects, there has been proposed a method for preventing or treating osteoporosis, which can minimize bone loss and promote bone formation by using natural materials such as foods and medicinal plants that are low in toxicity and can be consumed for food in daily life Application studies have been variously tried. (Kwak et al., 2008, Tsai et al., 2008). In this study, we investigated the effects of osteoclast-induced osteoclast differentiation on osteoclast- .

Accordingly, the inventors of the present invention have found that when using a composition containing nutrients and ginseng extract as an active ingredient while studying natural materials such as foods and medicinal plants that are low in toxicity and can be consumed for food in daily life in order to minimize side effects, The inventors of the present invention have found that it is possible to prevent the cytotoxicity of the extract of high concentration of ginseng compared with the case of using each herbal medicine extract alone.

It is an object of the present invention to provide an osteoclast and an osteoclast differentiating effect of osteoblast and osteoclast differentiation as an effective ingredient to provide an osteoclast activity inhibiting effect, an effect of increasing bone density and bone content, The present invention relates to a pharmaceutical composition for prevention and treatment of osteoporosis, which has the effect of preventing the cytotoxicity of high-concentration ginseng extract by mixing them with each other, And a health functional food composition for prevention and improvement of osteoporosis.

To this end, the present invention provides a pharmaceutical composition for prevention and treatment of osteoporosis, which comprises as an active ingredient a water extract and a ginseng extract, and the cytotoxicity of the ginseng extract is alleviated through a water extract.

In a preferred embodiment of the present invention, 10 to 900 parts by weight of ginseng extract may be added to 100 parts by weight of the extract.

In a preferred embodiment of the present invention, the concentration of the ginseng extract may be 10 to 70 占 퐂 / ml.

In a preferred embodiment of the present invention, when the concentration of the ginseng extract is 25 to 50 占 퐂 / ml, the survival rate of the osteoclast treated with the composition containing 100 parts by weight of the extract of ginseng And TRAP activity can be reduced by 65 ~ 75% compared to the untreated group.

In a preferred embodiment of the present invention, when the concentration of the ginseng extract is 25 to 50 占 퐂 / ml, the survival rate of the osteoblasts treated with the composition containing 100 parts by weight of the extract of ginseng When the concentration of the ginseng extract is 25 to 30 占 퐂 / ml, calcined nodules of osteoblasts treated with a composition containing 100 parts by weight of ginseng extract against 100 parts by weight of the extract of ryegrass It can be improved by 25 ~ 30% compared to the untreated group.

Another aspect of the present invention provides a health functional food composition for prevention and improvement of osteoporosis, which comprises as an active ingredient a water extract and a ginseng extract, wherein cytotoxicity caused by the ginseng extract is alleviated through a raspberry extract.

In a preferred embodiment of the present invention, when the concentration of the ginseng extract is 25 to 50 占 퐂 / ml, the survival rate of the osteoblasts treated with the composition containing 100 parts by weight of the extract of ginseng Treated with a composition comprising 100 parts by weight of ginseng extract against 100 parts by weight of the extract of ryegrass when the concentration of the ginseng extract is 25 to 30 占 퐂 / Can be improved by 25 ~ 30% compared with the untreated group.

According to the present invention, there is exhibited a combined effect that both the effect of increasing the bone density and the bone content and the effect of densifying the microstructure of the bone can be seen at the same time. In particular, when 100 to 400 parts by weight of ginseng extract is contained in 100 parts by weight of the extract, 100 to 400 parts by weight of ginseng extract is preferably used in 100 parts by weight of the extract, Can inhibit the cytotoxicity of the high concentration ginseng extract, and thus can be widely used as medicines or health foods useful for the prevention and treatment of osteoporosis.

1 is a graph showing the cytotoxicity of MTT assay of the composition for preventing or treating osteoporosis according to the present invention.
2 is a graph showing the TRAP activity evaluation of the composition for preventing or treating osteoporosis according to the present invention.
FIG. 3 is an image of TRAP-positive cells of osteoclasts induced by RANKL.
FIG. 4 is a graph showing the number of round-shaped osteoclasts (ROC) according to a specific mixing ratio of the extract of rumen extract and ginseng extract.
FIG. 5 is a graph showing cell survival rate and TRAP activity according to mixing ratios of various concentrations of water extract and ginseng extract.
FIG. 6 is an image and graph for evaluating the genetic factor in the absorption step of osteoclast induced by RANKL.
FIG. 7 is a graph showing the survival rate of osteoblast cells according to the mixing ratios of various concentrations of the water extract and ginseng extract.
8 is a graph showing cell survival rate and formation of calcified nodules of osteoblasts derived from MC3T3-E1 cells.

The present invention relates to a pharmaceutical composition for preventing and treating osteoporosis or a composition for preventing and / or improving osteoporosis, wherein the cytotoxicity caused by the ginseng extract is alleviated through the extract of raspberry extract, By providing the composition, it is possible to minimize osteoporosis and promote bone formation by using extracts of wax and ginseng, which are natural materials having excellent drug efficacy, without toxicity and side effects even when the drug is administered for a long time. Hereinafter, the present invention will be described in detail.

The Achyranthes bidentata Blume (AB) is a root of perennial herbaceous plants belonging to the genus Bifidum, which contains triterpenoid saponin, oleanolic acid, linoleic acid, serin, flavonoid ) And a large amount of calcium salt and is a natural product having analgesic, anti-inflammatory, antioxidant, anti-cancer and osteogenesis promoting effect and is not particularly limited as long as it can be harvested and / It is preferable to use one or more kinds of extracts selected from the group consisting of roots, stems and leaves.

At this time, it is preferable that the water extract is prepared using water, an alcohol having 1 to 5 carbon atoms, or a mixed solvent thereof as an extraction solvent, and more preferably, water is used as an extraction solvent.

In addition, the ginseng (Panax ginseng CA Meyer, PG) has various effects ranging from the prevention of osteoporosis including the enhancement of immune function, anticancer, sexual function, etc. Especially, ginsenoside Rh2 of saponin- And is not particularly limited as long as it can be usually harvested and / or purchased, and its form is not particularly limited. However, one or more kinds of extracts selected from the group consisting of roots, stems and leaves of ginseng are used .

At this time, the ginseng extract is preferably prepared using water, an alcohol having 1 to 5 carbon atoms, or a mixed solvent thereof as an extraction solvent, and more preferably, water is used as an extraction solvent.

The pharmaceutical composition for the prevention and treatment of osteoporosis and the health functional food composition for osteoporosis prevention and improvement according to the present invention preferably contain 10 to 900 parts by weight of the ginseng extract per 100 parts by weight of the extract of the mist extract, And 100 to 400 parts by weight of the extract of ginseng with respect to 100 parts by weight of the dried extract.

Although the ginseng extract has an excellent inhibitory effect on osteoclast differentiation as the concentration is high, it has the effect of preventing and improving osteoporosis, but has a problem of showing cytotoxicity at a high concentration. Therefore, the use of the ginseng extract and the extract of raspberry extract can reduce the cytotoxicity of the ginseng extract due to the anti-inflammatory and antioxidative action of the raspberry extract.

When the ginseng extract is contained in an amount of less than 10 parts by weight with respect to 100 parts by weight of the extract, the content of ginseng extract inhibiting osteoclast differentiation and facilitating osteoblast differentiation activity is lowered and the effect of improving osteoporosis is lowered. There is a problem that the cytotoxicity of the ginseng extract is increased.

That is, the higher the content of ginseng extract contained in the extract, the more excellent osteoporosis can be improved and prevented, but the higher the content of ginseng extract, the lower the cell survival rate. Do.

In the pharmaceutical composition for prevention and treatment of osteoporosis and the health functional food composition for osteoporosis prevention and improvement according to the present invention, the concentration of the ginseng extract is preferably 10 to 70 占 퐂 / ml, more preferably 10 to 50 占 퐂 / Ml. The higher the concentration of the ginseng extract, the better osteoporosis can be obtained, but the cell viability may be lowered. When the concentration of the composition is less than 10 μg / ml, the osteoclast differentiation inhibiting ability and osteoblast differentiation activity are deteriorated. When the concentration of the composition is more than 100 μg / ml, the diacetylene derivative There is a problem that the cell viability is lowered due to the effect of cytotoxicity caused by the cytotoxicity.

In this case, when the concentration of the ginseng extract is 25 to 50 / / ml, the survival rate of the osteoclast treated with the composition containing 100 parts by weight of the extract of rush extract with 100 parts by weight of the extract of the rush extract is 1 ~ 15%, and TRAP activity can be reduced by 65 ~ 75% compared to the untreated group.

When the concentration of the ginseng extract is 25 to 50 占 퐂 / ml, the survival rate of the osteoblast treated with the composition containing 100 parts by weight of the ginseng extract against 100 parts by weight of the extract is 1 to 10% higher than that of the untreated group The calcined nodule of osteoblast treated with a composition containing 100 parts by weight of ginseng extract against 100 parts by weight of the extract of ryegrass was 25 ~ 30% higher than that of the untreated group when the concentration of the ginseng extract was 25 ~ 30 / / Can be improved.

Accordingly, the present invention provides a pharmaceutical composition comprising the pharmaceutical composition for preventing and treating osteoporosis. The composition according to the present invention may be administered orally or parenterally in various clinical formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, &Lt; / RTI > 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 ) Or 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 non-aqueous solvent and 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. In addition, calcium or vitamin D3 may be added for the purpose of preventing or treating osteoporosis.

The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, Kg, preferably 300 to 700 mg / kg, most preferably 150 to 450 mg / kg, and can be administered 1 to 6 times a day.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of osteoporosis.

In addition, the health functional food composition for prevention and improvement of osteoporosis, which comprises the extract of rumen ginseng and ginseng extract according to the present invention effectively inhibits osteoclast differentiation and increases the differentiation activity of osteoblast, , The present invention provides a health functional food comprising the above food composition for preventing or improving osteoporosis. When the composition according to the present invention is used as a food additive, the mixed extract may be directly added or used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment).

However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range Is sure.

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

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides a food composition for prevention and improvement of osteoporosis, which comprises an extract of rubella and a ginseng extract as an active ingredient. The composition comprising the extract of rumen extract and ginseng extract according to the present invention effectively inhibits the differentiation of osteoclasts and increases the differentiation activity of osteoblasts and thus is effective for prevention or improvement of osteoporosis and thus is useful as a food composition for preventing or ameliorating osteoporosis Can be used.

The present invention provides a health functional food comprising the food composition for preventing or ameliorating osteoporosis. When the composition according to the present invention is used as a food additive, the mixed extract may be directly added or used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range Is sure.

Hereinafter, preferred embodiments and experimental examples are provided to facilitate understanding of the present invention. However, the following examples and experimental examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

[ Example ]

Manufacturing example One. The Preparation of extract

The wax was purchased from Korea Medicine Herbal Association. It was washed thoroughly with water, and the dried wax was cut at regular intervals and introduced at a rate of 100 g per 1 L of water. , Which was cooled and filtered at room temperature. The extract was concentrated using a rotary evaporator (Rotavapor RII, Buchi, Switzerland) and lyophilized (Martin Christ, Osterode Germany) in powder form and stored at 20 ° C. At this time, the final yield was 24.8%.

Manufacturing example 2. Preparation of ginseng extract

Ginseng was purchased from the Korea Medicine Herbal Association. The 4-year-old ginseng root was removed at regular intervals and introduced at a rate of 100 g per 1 L of water. , Which was cooled and filtered at room temperature. The extract was concentrated using a rotary evaporator (Rotavapor RII, Buchi, Switzerland) and lyophilized (Martin Christ, Osterode Germany) in powder form and stored at 20 ° C. At this time, the final yield was 33.3% of ginseng.

Example 1 ~ 8. Preparation of composition for the treatment and prevention of osteoporosis

The water extracts prepared in Preparation Example 1 and the ginseng extracts prepared in Preparation Example 2 were dissolved in DPBS (Dulbecco's phosphate buffered saline, Daegu, Korea) at a concentration of 100 mg / ml, Thereby preparing a composition for treating and preventing osteoporosis according to the present invention.

Threaded (1: 1 mixed) Concentration of water extract
(Unit: 占 퐂 / ml) Concentration of ginseng extract
(Unit: 占 퐂 / ml) The concentration of the mixed composition
(Unit: 占 퐂 / ml) Example 1 0 0 0 Example 2 20 20 10 Example 3 50 50 25 Example 4 100 100 50 Example 5 200 200 100 Example 6 500 500 250 Example 7 1000 1000 500 Example 8 2000 2000 1000

Example 9 ~ 17. Preparation of composition for the treatment and prevention of osteoporosis

The raspberry extract prepared in Preparation Example 1 and the ginseng extract prepared in Preparation Example 2 were dissolved in DPBS (Dulbecco's phosphate buffered saline, Daegu, Korea) at a concentration of 100 μg / ml, To prepare a composition for treating and preventing osteoporosis.

Raspberry extract
(Unit: μl) Ginseng extract
(Unit: μl) ratio Example 9 900 100 9: 1 Example 10 800 200 8: 2 Example 11 700 300 7: 3 Example 12 600 400 6: 4 Example 13 500 500 5: 5 Example 14 400 600 4: 6 Example 15 300 700 3: 7 Example 16 200 800 2: 8 Example 17 100 900 1: 9

Comparative Example 1 ~ 16. Preparation of composition for treating and preventing osteoporosis

The rumen extract prepared in Preparation Example 1 and the ginseng extract prepared in Preparation Example 2 were dissolved in DPBS (Dulbecco's phosphate buffered saline, Daegu, Korea) at a concentration of 100 mg / ml and diluted to the concentration shown in Table 3 Thereby preparing a composition for treating and preventing osteoporosis according to the present invention.

Concentration of wheat extract
(Unit: 占 퐂 / ml) Ginseng extract concentration
(Unit: 占 퐂 / ml) Comparative Example 1 0 - Comparative Example 2 10 - Comparative Example 3 25 - Comparative Example 4 50 - Comparative Example 5 100 - Comparative Example 6 250 - Comparative Example 7 500 - Comparative Example 8 1000 - Comparative Example 9 - 0 Comparative Example 10 - 10 Comparative Example 11 - 25 Comparative Example 12 - 50 Comparative Example 13 - 100 Comparative Example 14 - 250 Comparative Example 15 - 500 Comparative Example 16 - 1000

[ Experimental Example ]

All of the experimental groups described in the following Experimental Examples were performed in three or more groups, and the quantitative results were expressed as a percentage of the control group as the mean ± standard deviation. All experiments were repeated 3 times or more and used as the same test results. Two-tailed Student's t test was used to analyze the statistical significance, and p <0.05 was considered statistically significant.

Experimental Example 1. Measurement of osteoclast survival rate and evaluation of toxicity

MTT assay was performed to evaluate the survival and indirect toxicity of the composition of the present invention to osteoclasts. Mouse macrophage RAW 264.7 cells were seeded at a density of 1 × 10 ⁴ cells / well in a 96-well plate and stabilized for 24 hours in DMEM with 10% FBS and 1% penicillin-streptomycin. 50 ng / ml of RANKL (PeproTech EC (London, England)) was added to α-MEM (Gaithersburg, MD) containing 10% FBS (Gaithersburg, MD) and 1% penicillin-streptomycin for induction of osteoclast differentiation And then treated with the compositions of Examples 1 to 8 and Comparative Examples 1 to 16 above.

At that time, the group not treated with the composition was set as a control group. After 3 days of incubation, MTT solution (2 mg) was added, and the cells were incubated in an incubator (apparatus) at 5% CO ₂ and 37 ° C for 2 hours. formazan crystals were dissolved in DMSO. The absorbance was confirmed at 540 nm using an ELISA reader (BIO-RAD 450, USA). The results are shown in Table 4 and FIG. Here, a in FIG. 1 is Comparative Examples 1 to 8, b is Comparative Examples 9 to 16, and c means Examples 1 to 8.

density
(占 퐂 / ml) 0 10 25 50 100 250 500 1000 Whey extract (a) 100 99.90403 101.4395 97.12092 86.61228 82.58157 81.046007 77.30326 Ginseng extract (b) 100 107.9586 106.295 84.84712 52.29317 28.86691 24.32554 20.95324 Mixed extract (c) 100 106.2073 112.6249 107.575 62.01999 33.40347 31.0363 25.6707

According to Table 4 and Fig. 1, the treatment groups of Examples 1 to 8 and Comparative Examples 1 to 16 showed significantly lower survival rates than the control group at concentrations of 100 占 퐂 / ml and higher.

In particular, ginseng exhibited a low survival rate (84.85%) at a concentration of 50 μg / ml (Comparative Example 12), but no statistical significance and was significantly less than 52.93% at concentrations of 100 μg / And low cell survival rate. This may be due to the effect of cytotoxicity of diacetylene derivatives such as panaxydol, panaxinol, panaxitiol and acetyl panaxydol, which are known as anticancer substances in the fat soluble fraction of ginseng.

On the other hand, in the case of Examples 1 to 8 in which the husk and ginseng extracts were mixed at the same ratio, the cytotoxicity was shown to be 62.02% at the concentration of 100 ㎍ / ㎖ (Example 5) (107.58%) than the concentration of 50 占 퐂 / ml of the group treated with only the ginseng extract (Comparative Example 12) at a concentration of 50 占 퐂 / ml (Example 4) .

Therefore, it can be confirmed that the ginseng has cell cytotoxicity at a high concentration of 100 / / ml or more, but the cell survival rate is increased in the compositions of Examples 4 to 8. Thus, when the extract of ryegrass and ginseng is mixed, It is possible to reduce the cytotoxicity of ginseng extract due to anti-inflammatory and antioxidative cytoprotective action.

Experimental Example 2. TRAP Through dyeing Benign osteoclasts Differentiation Observation 1

TRAP solution assay was performed to measure TRAP activity of osteoclasts treated with the compositions of Examples 1 to 8 and Comparative Examples 1 to 16 according to the present invention. After the cells were washed with DPBS, 80 ㎕ of cold lysis buffer (90 mM citrate, pH 4.8, 0.1% Triton X-100 containing 80 mM sodium tartrate) was added to each well and treated at room temperature for 10 minutes. Substrate (20 mM p-nitrophenyl phosphate) was added to each well, followed by incubation in a 5% CO ₂ incubator at 37 ° C for 20 minutes. After stopping the reaction with 40 [mu] l of 0.5 N NaOH, the absorbance was confirmed at 405 nm using an ELISA reader, and the results are shown in Fig. 2 and Table 5 below. At this time, FIG. 2 (a) is Comparative Examples 1 to 8, b is Comparative Examples 9 to 16, and c means Examples 1 to 8.

density
(占 퐂 / ml) 0 10 25 50 100 250 500 1000 Whey extract (a) 100 97.47936 91.3371 90.36651 68.82515 56.10604 4.09503 32.34825 Ginseng extract (b) 100 40.31504 32.59906 19.96062 10.91558 6.743785 6.091558 6.608417 Mixed extract (c) 100 37.328 27.696 19.504 11.12 9.232 8.096 7.872

The TRAP enzyme has high activity when ATP and nitrophenyl phosphate are present and can be used to confirm osteoclast differentiation level by measuring activity of osteoclast during bone resorption.

According to FIG. 2, the TRAP activity of the wheat extract was not significantly different from that of the control group regardless of the concentration at 100 μg / ml or less (Comparative Examples 1 to 4), and the TRAP activity of 100 μg / And the activity was low at the concentration. However, it is believed that this is due to the reduced cell viability as confirmed in Experimental Example 1 above.

On the other hand, when treated with the ginseng extract, the TRAP activity was remarkably increased to 40.31%, 32.60%, and 19.96% at 10 (Comparative Example 10), 25 (Comparative Example 11) and 50 (Comparative Example 12) (Example 2) (37.33%), 25 (Example 3), and 50 (Example 4) 쨉 g / ml (19.51 %) In a concentration-dependent manner.

Therefore, although the extract of ranch does not affect the osteoclast differentiation induced by RANKL, it can be understood that the ginseng extract is directly involved in the inhibition of osteoclast differentiation in a concentration-dependent manner. That is, it can be confirmed that the extract of raspberry does not inhibit osteoclast differentiation.

Experimental Example 3. TRAP Through dyeing Benign osteoclasts Differentiation observation 2

In order to observe the differentiation of benign osteoclasts treated with the compositions of Examples 3 to 4 and Comparative Examples 3 to 4 and 11 to 12 according to the present invention, TRAP (tartrate) in the osteoclast cells induced from mouse macrophage RAW 264.7 cells resistant acid phosphate staining. Cells treated with differentiation agent and 3 days after treatment were washed with PBS, fixed with 10% formalin for 5 minutes, and washed again with purified water (DW). Sigma TRAP staining (Sigma-Aldrich kit no. 387) was treated with fresh TRAP solution for 30 minutes according to instructions for use. The stained cells were observed with an optical microscope (CKX41, Olympus, Japan), images were imaged with a digital image camera (DIXI 3000, China), and the results are shown in Fig. In Examples 3 to 4 and Comparative Examples 3 to 4 and 11 to 12 according to the present invention, as shown in Experimental Examples 1 and 2, there was no lethal activity by the extract of rumen extract and ginseng extract, and low TRAP activity Treated with a composition having a visible concentration of 25 and 50 / / ml, cultured for 3 days, and then subjected to TRAP staining.

3A to 3I are as shown in Table 6 below, and arrows indicate a round shape wave

Bone cells.

a b c Control group Comparative Example 3 (25 ug / ml of mist) Comparative Example 4 (50 占 퐂 / ml of mist) d e f Control group Comparative Example 11 (25 g / ml of ginseng) Comparative Example 12 (Ginseng 50 占 퐂 / ml) g h i Control group Example 3 (equivalent amount of 25 [mu] g / ml) Example 4 (equivalent amount 50 쨉 g / ml)

According to FIG. 3, TRAP-positive polynuclear osteoclasts, which have a round-shaped osteoclast (ROC), that is, a bone-absorbing osteoclast formed by the formation of polynuclear mature osteoclasts, . The osteoclast differentiation pattern similar to that of the control group was observed in the 25 and 50 ㎍ / ㎖ treated groups (Comparative Examples 3 and 4), whereas the osteoclast treated group and the mixed oyster and ginseng treated groups showed significantly different osteoclast differentiation Abnormal ROC and unfused mononuclear proliferating osteoclasts and irregular shaped osteoclasts (IOCs) were abundantly observed.

Experimental Example 4. TRAP Through dyeing Benign osteoclasts Differentiation inhibition 3

In order to evaluate the TRAP activity of the osteoclasts treated with the compositions of Examples 3 and 4 and Comparative Examples 3, 4, 11 and 12 according to the present invention, TRAP (tartrate) in osteoclast cells induced from mouse macrophage RAW 264.7 cells resistant acid phosphate staining. After 3 days, the cells were fixed with DPBS and fixed with 10% formalin for 5 minutes and rinsed again with DW. Sigma TRAP staining (Sigma-Aldrich kit no. 387) was treated with fresh TRAP solution for 30 minutes according to instructions for use. The stained cells were observed with an optical microscope (CKX41, Olympus, Japan), imaged with a digital imaging camera (DIXI 3000, China) to measure the number of round-shaped osteoclasts (ROC) The results are shown in Fig. 4 and Table 7 below.

Concentration (/ / ml) 0 25 50 Raspberry extract 100 106.2857 102.2857 Ginseng extract 100 42.51497 42.51497 Mixed extract 100 37.86982 27.21893

According to FIG. 4, the number of round-shaped osteoclasts (ROC) does not change with the concentration of the wheat extract, but the number decreases when the ginseng extract is contained at 25 and 50 ㎍ / ㎖ And that the degree of decrease was greater at higher concentrations, so that the number of round osteoclasts was significantly decreased in a concentration-dependent manner.

Experimental Example 5. TRAP Through dyeing Benign osteoclasts Differentiation inhibition 3

To observe the differentiation and TRAP activity of benign osteoclasts treated with the compositions of Examples 9 to 17 according to the present invention, cytotoxicity and TRAP activity were measured in the same manner as in Experimental Examples 1 and 2, Are shown in Table 8 and FIG.

Control group Example 9 Example 10 Example 11 Example 12 Example 13 Example 14 Example 15 Example 16 Example 17 cell
toxicity 100 117.459 109.8539 107.0716 109.599 106.6079 94.92233 88.361 85.880 80.66311 TRAP
activation 100 101.6581 83.70497 66.72384 55.3745 47.4271 45.82619 43.71069 37.10692 32.53288

According to FIG. 5, the cytotoxicity of the mixed extracts of rumen extract and ginseng extract was not significantly decreased. At this time, TRAP activity tended to decrease significantly as the content of ginseng extract increased.

On the other hand, it can be seen that the rate of decrease of TRAP activity is smaller than that of the group treated with the composition prepared by mixing the same amount of the wheat extract and ginseng extract (Example 5).

Experimental Example 6. Reverse transcription polymerase chain reaction ( RT - PCR ) analysis

In order to examine the expression of calcitonin receptor (Cal-R) and TRAP in osteoclast treated with the compositions of Examples 3 and 4 and Comparative Examples 3, 4, 11 and 12 according to the present invention, RNA was extracted and cDNA was synthesized with iScript cDNA synthesis kit. The polymerase chain reaction (PCR) was carried out at 95 ° C for 10 minutes using Emerald Taq, followed by 1 minute at 95 ° C, 30 seconds at 51 ~ 58 ° C, and 1 minute at 72 ° C. After 25-32 cycles, final amplification was performed at 72 ° C for 10 minutes. The order of PCR primers used is shown in Table 9 below. The reacted samples were electrophoresed on 2% agarose gel, stained with Et-Br, observed on UV, and digitized using densitometric analysis. The results are shown in FIGS. 6 and 10.

Gene Primer sequence (5'-3 ') Annealing
Temp (캜) Product size (bp) Cal-R
F-ACCGACGAGCAACGCCTACGC 58
490
R-GCCTTCACAGCCTTACAGGTAC TRAP F-AAATCACTCTTTAAGACCAG 51
317
R-TTATTGAATAGCAGTGACAG GAPDH
F-AACTTTGGCATTGTGGAAG 53
223
R-ACACATTGGGGGTAGGAACA

Cal-R TRAP Concentration (/ / ml) 0 25 50 0 25 50 Raspberry extract 100 82.285 90.79786 100 99.05167 98.24505 Ginseng extract 100 36.73364 15.23028 100 68.34427 58.75493 Mixed extract 100 49.04299 35.56758 100 96.28895 61.34352

Osteoclasts are formed by complex processes such as initiation, differentiation, polynucleation, and maturation of various cytokines, hormones and genetic factors. There are abundant calcitonin receptors (Cal-R) and TRAP, , And it is known that it absorbs the bone matrix by forming an acting ring.

According to the results shown in Table 10 and FIG. 6, the mRNA expression level of Cal-R in the bone morphogenesis stage of mature osteoclasts was analyzed. As a result, the mushroom extract-treated group (Comparative Examples 3 and 4) Did not appear. The expression level of Cal-R in the ginseng extract-treated group was 63.27% and 84.77% in a concentration-dependent manner at 25 μg / ml (Comparative Example 11) and 50 μg / ml (Comparative Example 12) have. It can be confirmed that the compositions prepared by mixing equal amounts of the wheat extract and the ginseng extract also show a reduction rate of 50.96% and 64.43% at 25 占 퐂 / ml (Example 3) and 50 占 퐂 / ml (Example 4), respectively. Significantly decreased expression rates at all concentrations and at a concentration of 50 μg / ml compared to 25 μg / ml were also confirmed.

In addition, as a result of analysis of the change in the amount of TRAP expression, the reduction rate was 31.66% and 41.25% at 25 ㎍ / ㎖ of the ginseng extract (Comparative Example 11) and 50 ㎍ / ㎖ (Comparative Example 12) Respectively. In the composition prepared by mixing the ascidian extract and ginseng extract in the same amount, it was confirmed that the composition showed a decrease of 38.66% only at the concentration of 50 / / ㎖ (Example 4) compared to the control, and the concentration of 25 / / Which is a decrease of 34.95%.

Therefore, it was found that the expression of mRNA of Cal-R and TRAP was significantly inhibited by ginseng extract as well as the inhibition of TRAP enzyme activity by RANKL. This suggests that ginsenoside Rb1 contained in ginseng extract regulates NF-kB and MAPKs pathways, and ginsenoside Rh2 regulates NF-kB, NFATc1 and c-Fos to inhibit osteoclast differentiation have. Therefore, it is expected that ginseng inhibited osteoclast differentiation transcription factor NFATc1 to reduce Cal-R and TRAP, and ultimately bone morphogenesis could be inhibited.

Experimental Example 7. Osteoblast differentiation effect

MC3T3-E1 cells were cultured in 96-well plates (8 × 10 ³ cells / well) with 10% FBS and 1% penicillin-streptomycin added to the osteoblasts to evaluate osteoblast survival and indirect toxicity. The cells were placed in MEM medium to stabilize the cells for 24 hours. Osteoblast differentiation agents were added to the same medium, treated with Examples 9 to 17 and Comparative Examples 3, 4, 11 and 12 according to the present invention, and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours MTT assay was performed to measure survival rate. At this time, the osteoblast differentiation agent includes OS, 1M 2-glycerophosphate, 50 mM ascorbic acid, and 10 mM dexamethasone.

Example 9 Example 10 Example 11 Example 12 Example 13 Example 14 Example 15 Example 16 Example 17 The 0 100 90 80 70 60 50 40 30 20 10 0 Ginseng 0 0 10 20 30 40 50 60 70 80 90 100 Osteoclast survival rate 100 100.5666 105.0992 104.3059 106.0623 101.6431 103.8527 99.2068 102.7762 100.4533 103.9093 103.6827

Concentration (/ / ml) 0 25 50 Raspberry extract 100 104.828 98.72364 Ginseng extract 100 98.02772 103.7846 Mixed composition 100 108.4437 102.3179

MC3T3-E1 cells, which are similar to osteoblasts existing in bone tissues, are used for the accumulation of bone matrix, calcification and growth factor metabolism, and the cytotoxicity of cells by proliferation and cell viability The osteoblast differentiation stage activity was confirmed, and the results are shown in Figs. 7, 8 (a), 10 and 11. The cell survival rate after treatment for 24 hours at the concentrations of all the experimental groups mixed with the same amount and the same mixing ratio of the various concentrations of the masks and ginseng of Examples 9 to 17 and Comparative Examples 3, 4, 11 and 12 according to the present invention . &Lt; / RTI >

Experimental Example 8. Calcification of nodule formation

Calcified nodules of osteoblast differentiated from MC3T3-E1 cells were measured using alizarin red-sulfate (AR-S). Cells were maintained in 24-well plates (2 × 10 4 cells / well) in serum-MEM supplemented with 10% FBS and 1% penicillin-streptomycin for 24 hours. The osteoblast-induced differentiation agent and the cells of Examples 3 and 4 and Comparative Examples 1, 3, 4, 9, 11, and 12 according to the present invention at concentrations of 0, 25 and 50 / / ml were cultured for 21 days. Cells were fixed with ice-cold 4% paraformaldehyde for 30 min at 4 ° C and stained with 40 mM AR-S staining solution for 10 min at room temperature. After washing with purified water (DW), the dye was reacted with 10% (w / v) cetylpyridinium chloride / 10 mM sodium phosphate (pH 7.0) for 15 minutes and the absorbance was confirmed at 562 nm using an ELISA reader , And the results are shown in Fig. 8 (b) and Table 13.

Concentration (/ / ml) 0 25 50 Raspberry extract 100 116.8399 106.237 Ginseng extract 100 125.9594 106.3205 Mixed composition 100 126.4113 94.35484

8 (b) and Table 12, the absorbance was measured by dissolving the calcified nodule stained with alizarin red. As a result, it was found that the ginseng extract of 25 / / ml (Comparative Example 11) 25 [mu] g / ml (Example 3) showed a significant increase of 25.96% and 26.41% compared with the control group. Wistar also showed an increasing tendency but did not show statistical significance.

Thus, it can be seen that the increase in osteoblast differentiation activity is apparent in the treatment group containing ginseng extract. This suggests that the ginsenoside ginsenoside component was affected by ginseng saponin.

Claims

As the active ingredient,
Wherein the cytotoxicity of the ginseng extract is alleviated through the extract of the extract.

The pharmaceutical composition for preventing and treating osteoporosis according to claim 1, wherein the extract comprises 10 to 900 parts by weight of ginseng extract per 100 parts by weight of the extract.

The pharmaceutical composition for preventing and treating osteoporosis according to claim 1, wherein the concentration of the ginseng extract is 10 to 70 占 퐂 / ml.

The method according to claim 1, wherein, when the concentration of the ginseng extract is 25 to 50 占 퐂 / ml, the survival rate of the osteoclast treated with the composition comprising 100 parts by weight of the ginseng extract against 100 parts by weight of the extract is 1 To 15%, and the TRAP activity is reduced by 65 to 75% as compared to the untreated group.

The method according to claim 1, wherein, when the concentration of the ginseng extract is 25 to 50 占 퐂 / ml, the survival rate of the osteoblast treated with the composition comprising 100 parts by weight of the ginseng extract against 100 parts by weight of the extract is 1 ~ 10%
When the concentration of the ginseng extract is 25 to 30 占 퐂 / ml, calcified nodules of osteoblast treated with a composition containing 100 parts by weight of ginseng extract against 100 parts by weight of the extract of ryegrass are improved by 25 to 30% Or a pharmaceutically acceptable salt thereof.

As the active ingredient,
Wherein the cytotoxic effect of the ginseng extract is alleviated through the extract of rats.

The health functional food composition according to claim 6, wherein the extract comprises 10 to 900 parts by weight of ginseng extract per 100 parts by weight of the extract.

The health functional food composition according to claim 6, wherein the concentration of the ginseng extract is 10 to 70 占 퐂 / ml.

7. The method according to claim 6, wherein when the concentration of the ginseng extract is 25 to 50 占 퐂 / ml, the survival rate of the osteoclast treated with the composition comprising 100 parts by weight of the ginseng extract against 100 parts by weight of the extract is 1 To 10%, and the TRAP activity is reduced by 65 to 75% as compared to the untreated group.

The method according to claim 6, wherein, when the concentration of the ginseng extract is 25 to 50 占 퐂 / ml, the survival rate of the osteoblast treated with the composition comprising 100 parts by weight of the ginseng extract against 100 parts by weight of the extract is 1 ~ 10%
When the concentration of the ginseng extract is 25 ~ 30 占 퐂 / ml, the calcified nodules of the osteoblast treated with the composition containing 100 parts by weight of the ginseng extract against 100 parts by weight of the raspberry extract are improved by 25 ~ 30% Wherein the health functional food composition for preventing and improving osteoporosis is a composition for preventing and improving osteoporosis.