KR20130060837A - A composition comprising the extract of processed panax genus plant for treating and preventing osteoporosis - Google Patents

Abstract

PURPOSE: A processed ginseng extract is provided to suppress expression of genes related to RANKL-induced osteoclast differentiation and bone resorption, and to prevent and treat metabolic bone diseases. CONSTITUTION: A pharmaceutical composition for preventing and treating metabolic bone diseases contains a processed Panax sp. plant extract in which the content of ginsenoside Rg3, Rk1, and Rg5 is higher than the content of ginsenoside Rb1, Rb2, RC, and Rd. The Panax sp. plant is artificially processed using Panax ginseng, Panax quinquefolia, Panax notoginseng, Panax vietnamensis, Panax japonicus, Panax elegatior, Panax wangianus, Panax bipinnatifidus, or Panax pseudoginseng. The Panax sp. plant is processed by thermal treatment of ginseng at 120-180 deg. C for 0.5-20 hours to enhance drug efficiency.

Description

A composition comprising the extract of processed Panax genus plant for treating and preventing osteoporosis}

It relates to a pharmaceutical composition for the treatment and prevention of bone metabolic diseases containing the processed ginseng extract of the present invention as an active ingredient.

Kumamoto H, Ooya K. ExpreASion of parathyroid hormone-related protein (PTHrP), osteoclast differentiation factor (ODF) / receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoclastogenesis inhibitory factor (OCIF) / osteoprotegerin (OPG) in ameloblastomas. J Oral Pathol Med. 2004; 33 (1): 46-52.

2. Reddy SV. Regulatory mechanisms operative in osteoclasts. Crit Rev Eukaryot Gene Expr. 2004; 14 (4): 255-70.

3.Pang M, Martinez AF, Fernandez I, Balkan W, Troen BR. AP-1 stimulates the cathepsin K promoter in RAW 264.7 cells. Gene. 2007; 403 (1-2): 151-8.

Boyce BF, Yamashita T, Yao Z, Zhang Q, Li F, Xing L. Roles for NF-kappa B and c-Fos in osteoclasts. J Bone Miner Metab. 2005; 23 Suppl: 11-5.

Khosla S. Minireview: the OPG / RANKL / RANK system. Endocrinology. 2001; 142 (12): 5050-5.

6.Han SY, Lee NK, Kim KH, Jang IW, Yim M, Kim JH, Lee WJ, Lee SY. Transcriptional induction of cyclooxygenase-2 in osteoclast precursors is involved in RANKL-induced osteoclastogenesis. Blood. 2005; 106 (4): 1240-5.

7.Yamashita M, Otsuka F, Mukai T, Yamanaka R, Otani H, Matsumoto Y, Nakamura E, Takano M, Sada KE, Makino H. Simvastatin inhibits osteoclast differentiation induced by bone morphogenetic protein-2 and RANKL through regulating MAPK, AKT and Src signaling. Regul Pept. 2010; 162 (1-3): 99-108

Murakami A, Song M, Ohigashi H. Phenethyl isothiocyanate suppresses receptor activator of NF-kappaB ligand (RANKL) -induced osteoclastogenesis by blocking activation of ERK1 / 2 and p38 MAPK in RAW264.7 macrophages. Biofactors. 2007; 30 (1): 1-11.

9.H Choi HJ, Park YR, Nepal M, Choi BY, Cho NP, Choi SH, Heo SR, Kim HS, Yang MS, Soh Y. Inhibition of osteoclastogenic differentiation by Ikarisoside A in RAW 264.7 cells via JNK and NF-kappaB signaling pathways. Eur J Pharmacol. 2010; 636 (1-3): 28-35

10. Understanding Korean Ginseng, Korean Ginseng Society, p 9, 1995; Advances in Ginseng Research, Korea Ginseng Society, p 127, 1998)

11. Understanding Korean Ginseng, Korean Ginseng Society, pp 9-18, 2008).

12. Kim W.Y. et al., Journal of Natural Products, vol. 63, pp 1702-1704, 2000;

13 Kwon S W, et al., J. Chromatogr. A, 921 (2), pp 335-339, 2001;

14.Yong-Sam Keum, Kwang-Kyun Park, Jong-Min Lee, Kyung-soo Chun., Antioxidant and anti-tumor promoting activities of the methanol extract of heat-processed ginseng. J. CancerLetters . 150, pp. 41-48,2000.

15. Young C. Kim, So R. Kim, George J. Markelonis, Tae H. Oh., Ginsenosides Rb1 and Rg3 Protect Cultured Rat Cortical Cells From Glutamate-Induced Neurodegeneration. J. Neuroscience Res . 53, pp. 426-432, 1998.

16. Keun Young Jung, Dong seon Kim, Platelet Activating Factor Antagonist Activity of Ginsenosides. Biol . Pharm . Bull . 21.PP.79-80, 1998.

17. Kim WY, et al., J. Nat . Prod . , 63 (12) , pp 1702-1704, 2001;

18. Kwon SW, et al., J. Chromatogr . A. , 921 (2) , pp 335-339, 2001

19. Republic of Korea Patent 10-0192678 Processed Ginseng Product with Enhanced Drug Effect, 1999

It relates to a pharmaceutical composition for the treatment and prevention of bone metabolic diseases containing the processed ginseng extract of the present invention as an active ingredient.

Metabolism is composed of osteoblasts responsible for bone formation and osteoclasts responsible for bone resorption, and the function of these cells is balanced to maintain homeostasis. However, when osteoblast activity decreases or osteoclast activity increases, bone density decreases and osteoporosis may be induced. Factors causing osteoporosis include menopause, hyperthyroidism, diabetes, stress, lack of smoking and exercise, physical aging, and the use of glucocorticoid drugs in women (1-3).

Osteoclasts are differentiated by various differentiation-causing factors in macrophage progenitor cells (4-5). In particular, RANKL (receptor activator of nuclear factor kappa B ligand) secreted from osteoblasts binds to osteoclast precursor cells and RANK (receptor activator of nuclear factor kappa B) present on the surface of osteoclasts. Causes differentiation and activation (6). Differentiated osteoclasts were activated by NF-B, c-Fos, c-jun, AP-1, NFATc1, MAPK, ERK, JNK, p38 activation process, Src, Akt MITF activation, etc. It promotes the expression of osteoclast-specific proteins such as cathepsin K and calcitonin receptor (7-9).

Panax is a perennial herbaceous plant belonging to the genus Araliaceae in plant taxonomy. Representative species include Korean ginseng ( Panax ginseng ), flower ginseng ( Panax quinquefolia ), ginseng (samchi, Panax notoginseng ), Vietnamese ginseng ( Panax vietnamensis ) and Panax japonicus (People's Understanding, Korean Ginseng Society, p 9, 1995; Advances in Ginseng Research, Korean Ginseng Society, p 127, 1998). Thior ( Panax elegatior ), Panax wanzianus ( Panax wangianus , Panax bipinnatifidus ), Panax pseudoinsins ( Panax pseudoginseng ).

Ginseng, one of the most famous Panax plants, is one of the most widely used herbal medicines in Asia. It has been widely used in Asia for many years. It has been widely used for many purposes such as fatigue, nourishment, improvement of cognitive function, memory, blood circulation improvement, And the inhibition of platelet aggregation are known. (Understanding of Korean Ginseng, Korean Ginseng Society, pp 9-18, 2008).

On the other hand, heat-processing or treating a genus of Panax plants with a weak acid removes some sugars and alcohol groups from the ginsenoside structure, and the new structure of ginsenosides having stronger potency, ie, ginsenosides Rg3, Rg5, Rk1, Rk2 , Rk3, Rs1, Rs2, Rs3, F4, Rh2, Rh3, Rh4 and the like are known to be produced (Kim WY et al, Journal of Natural Products, vol. 63, pp1702-1704, 2000; Kwon SW, et al. , J. Chromatogr.A, 921 (2), pp 335-339, 2001; Park IH et. Al., Archives of Pharmacal Research, 25, pp.428-432 and 837-841, 2002).

Republic of Korea Patent No. 192678 and U.S. Patent No. 5,76,460 include processed panax plants in which the sum of the contents of ginsenosides (Rg3 + Rg5 + Rk1) is greater than the sum of the contents of (Rb1 + Rb2 + Rc + Rd). Hereinafter, the ginseng) is described, wherein the ginseng is a processed ginseng by dramatically increasing the content of Rg ₃ , Rg ₅ , Rk ₁ , which is highly effective by treating ginseng ( Panaxginseng ) at high temperature and high pressure. As the medicinal effect of ginseng has been reported anti-cancer effect (14), brain function improving effect (15), platelet aggregation ability blocking effect (16) and the like.

However, none of the above documents has yet described or taught any osteoporosis treatment and prophylactic effects of the processed Panax genus extract.

The present inventors tried to evaluate the action of inhibiting RANKL induced osteoclast differentiation and bone resorption related gene expression using extracts of ginseng, processed ginseng. To this end, extracts of ginseng, processed ginseng, inhibited TRAP (+) multinucleogenesis in the cells differentiated into osteoclasts by human RANKL treatment and processed gene expressions such as TRAP, NFATc1, JNK, which are osteoclast related factors. Confirmed that the extract of ginseng ginseng inhibited to complete the present invention.

In order to solve the above object, the present invention is processed Panax genus plant extract in which the sum of the ginsenoside Rg3, Rk1, Rg5 content is higher than the sum of the content of ginsenoside Rb1, Rb2, Rc, Rd It provides a pharmaceutical composition for treating and preventing bone metabolic diseases and bone diseases containing as an active ingredient.

The processed Panax plant as defined herein is preferably Korean Ginseng (Panax ginseng), Firewood (Panax quinquefoliaHowever,Panax notoginseng), VietnamPanax vietnamensis),Panax japonicus), Panax Elegatior (Panax elegatior), Panax Wanian (Panax wangianus), Panax bisfinatipus (Panax bipinnatifidus), Or Panax Pseudogin (Panax pseudoginsengThe present invention relates to a processed ginseng which includes artificially processed plants), and more preferably, has enhanced efficacy by heating at a high temperature. More specifically, the ginseng is heated at a high temperature of 120 to 180 ° C. for 0.5 to 20 hours to increase the active ingredient so that the ratio of ginsenosides (Rg 3 + Rg 5) / (Rc + Rd + RbR + Rb₂) is 1.0 or more. Contains processed ginseng.

The extract is a lower alcohol such as water, methanol, ethanol, butanol, or a mixed solvent thereof, preferably water or 10 to 100% water and ethanol mixed solvent, more preferably, about 50 to 90% water and ethanol mixed Extracts soluble in the solvent.

The bone metabolic disease as defined herein includes osteoprosis, paget disease, periodontal disease, metastatic cancer or rheumatoid arthiritis, preferably osteoporosis. Characterized in that.

Hereinafter, the method of obtaining the extract of the present invention will be described in more detail.

For example, after washing and drying the fruits, stems or roots of the Panax plants, respectively, the Panax plants are heat treated at a high temperature of 120 to 180 ° C. for 0.5 to 20 hours and then about 1 times the weight of the individual samples. To 30 times, preferably about 5 to 15 times (w / v) volume of water, C ₁ to C ₄ lower alcohols or mixed solvents thereof, preferably water or ethanol, more preferably water or Heat extraction method, ultrasonic extraction method, reflux extraction method for about 1 to 6 hours, preferably 3 to 5 hours at a reaction temperature of about 70 to 120 ℃, preferably 80 to 110 ℃ using 50 to 90% ethanol as the extraction solvent A second step of repeated extraction 1 to 10 times, preferably 2 to 7 times by a conventional extraction method such as a reflux extraction method; The processed Extract of Phanax spp. Of the present invention can be obtained through the third step of filtration and concentration under reduced pressure of the extract obtained in the above step.

In another aspect, the present invention provides a pharmaceutical composition for treating and preventing bone metabolic diseases and bone diseases containing the above production method and processed Panax genus plant extract prepared by the production method as an active ingredient.

The extract prepared above was confirmed to have an effect of inhibiting RANKL-induced osteoclast differentiation and bone resorption-related gene expression, and in this study, the extract of ginseng, which is processed ginseng, in the cells differentiated into osteoclasts by RANKL treatment was TRAP (+ ) It has been shown to inhibit the production of multinucleated cells, and to inhibit the gene expression of osteoclast-related factors such as TRAP, NFATc1, JNK, etc., which can be useful for osteoporosis and bone diseases. The composition of the present invention comprises 0.1 to 50% by weight of the herbal extract based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

Compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract is preferably administered at a dose of 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Therefore, the dose is not intended to limit the scope of the present invention in any aspect.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, including, for example, oral and rectal, or intravenous.

As described above, it was confirmed that the processed ginseng extract of the present invention has an effect of inhibiting RANKL-induced osteoclast differentiation and bone resorption-related gene expression, and in this study, ginseng which is processed ginseng in cells differentiated into osteoclasts by RANKL treatment. Extracts showed TRAP (+) multinucleated cell suppression effect, and inhibited gene expression of osteoclast-related factors TTRAP, NFATc1, JNK, etc., for the treatment and treatment of bone metabolic diseases and bone diseases This can be useful.

1 is a diagram showing the effect on the formation of TRAP (+) multinucleated cells from RANKL treated RAW264.7 cells according to the concentration of processed ginseng extract (SG) of the present invention,
Figure 2 is a view showing the effect on TRAP expression according to the processed ginseng extract (SG) concentration of the present invention,
Figure 3 is a view showing the effect on the expression of NFATc1 according to the concentration of processed ginseng extract (SG) of the present invention,
Figure 4 is a diagram showing the effect on JNK expression according to the processed ginseng extract (SG) concentration of the present invention.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, it should be understood that the present invention is not limited thereto.

Example 1 Preparation of Processed Ginseng Extract

Methods, such as steam (Kim WY, et al, J. Nat Prod, 63 (12), pp 1702-1704, 2001;..... Kwon SW, et al, J. Chromatogr A., 921 (2), pp 335-339, 2001).

That is, dried ginseng (Gumsan ginseng market) was finely chopped, and heated using steam at 120 ° C. and 15 atm for 4 hours. 2 L of 80% ethanol was added to 1 kg of processed ginseng (hereinafter referred to as ginseng), and the extract obtained by reflux extraction for 4 hours or more in a water bath was concentrated under reduced pressure and freeze-dried to obtain about 300 g of an extract powder. In the experiment, the ginseng extract (hereinafter referred to as SG) was dissolved in a medium using DMSO (Sigma D-2650), and used after passing a filter paper having a pore size of 0.45 μm. (Rg3 + Rg5 + Rk1) / (Rb1 + Rb2 + Rc + Rd) = 6.1 of the ginseng extract used in the experiment.

Example 2. Analysis of Saponin Content of Sea Ginseng Extract

The ginseng saponin component contained in the processed ginseng prepared in Example 1 according to the present invention was analyzed by the following method.

Four 40 ml stainless steel vessels were added with 5 g of white ginseng and 5 ml of water, respectively, and then sealed and heated at 110 ° C. for 2 hours, 120 ° C. for 2 hours and 3 hours, and 130 ° C. for 2 hours. 5 g of each of the processed ginseng and the commercially produced white ginseng and red ginseng were taken, extracted three times with 100 ml of methanol, concentrated, suspended in water, and extracted three times with 100 ml of ether. The remaining aqueous layer was extracted three times with 100 mL of butanol, and then the butanol fraction was concentrated. → 80/20/15, detector: analyzed by ELSD (Evaporative light scattering detector). The measured results are shown in Table 1 below.

Saponin
Sample Rb ₁ Rb ₂ Rc Rd Rg ₃ Rg ₅ (Rg ₃ + Rg ₅₎ /
_{(Rc + Rd + Rb 1 +} Rb 2) 120/3 hour 10.82 6.91 8.52 7.64 28.01 14.01 1.24 120/2 hours 10.46 7.74 10.66 5.58 24.12 11.35 1.03 130/2 hours 4.06 3.44 3.98 3.82 21.00 16.17 2.43 110/2 hours 19.02 11.55 10.68 8.37 7.02 4.39 0.23 White ginseng 22.15 3.25 5.86 2.47 0.00 0.00 0.00 Red ginseng 30.11 9.69 12.45 1.76 1.05 1.05 0.01

As can be seen from the results shown in the above Table 1, the processed ginseng obtained by the heat treatment according to the present invention significantly increases the contents of Rg and Rg components, which are ginseng saponins which are almost or completely absent from fresh ginseng, white ginseng and red ginseng And exhibits excellent drug efficacy.

According to the results as described above, in order to more specifically confirm the change in the content of saponin components, in particular ginsenoside Rg and Rg according to the change in the heating temperature for ginseng, heating is 100 ℃, 110 ℃, 120 ℃, 130 ℃, Rg and Rg content of ginseng obtained by 2 hours at 150 ° C., 160 ° C., 180 ° C. and 200 ° C. was measured and compared with the content in ginseng (salt) that was not heated. The results are shown in Table 2 below.

Heating temperature
(time)
Kinds Untreated
(Fresh ginseng) 100
2 hours 110
2 hours 120
2 hours 130
2 hours 150
2 hours 160
2 hours 180
2 hours 200
2 hours Rg ₃ 0.00 0.02 0.08 0.17 0.86 0.44 0.45 0.35 0.23 Rg ₅ 0.00 0.02 0.05 0.08 0.44 0.53 0.56 0.48 0.38

Note) Each ingredient content is expressed as% of the amount of fresh ginseng used.

As seen above, the content of ginsenosides such as Rg and Rg is significantly increased in the case of processed ginseng heated at 120 to 180 ° C as in the present invention compared to the case of untreated ginseng or red ginseng (100 ° C heating). It can be seen that. (Rg3 + Rg5 + Rk1) / (Rb1 + Rb2 + Rc + Rd) = 6.1 of the ginseng extract used in the experiment.

Experimental Example  1. Cell Culture and Drug Treatment

The RAW 264.7 cells used in the experiment were cultured in a CO2 cell incubator using DMEM (Dulbecco's modified eagle medium) / 10% fetal bovine serum (FBS) / PC-SM medium, and the number of cells was 96 well plate at 5x10 ³ cells / well. And cultured using. After culturing for 24 hours, the culture medium was discarded, and the cells were cultured by exchanging with a-MEM to which 10% FBS, 50 ng / ml RANKL, and 1 ng / ml TGFb were added. Different concentrations of AS were added to the culture. It was incubated for 6 days while changing to the same medium every two days. The experimental group was administered with (1) RANKL-treated control group (N) (2) RANKL-induced control group (C), (2) RANKL-induced group + 1 / processed ginseng extract of the present invention of Example 1 (SG) (CM 0.1), (3) RANKL induction group + 5 / to the group (CM1) to which the processed ginseng extract (SG) of the present invention of Example 1 was administered.

Experimental Example 2. Measurement of osteoclast generation capacity

After inducing RAW 264.7 cells to osteoclasts with RANKL (receptor activator for nuclear factor B ligand), TRAP-positive multinucleated cells (TRAP (+) MNCs) were identified by staining TRAP, a marker for expression of mature osteoclasts. . Differentiated cells were washed twice with PBS, fixed with 3.7% formaldehyde-citrate-acetone solution for 10 minutes and washed twice with distilled water. Treat the cells prepared by mixing 2% TRAP fast garnet GBC base solution with NaNO3 solution in the same ratio and a solution containing 5% naphtha AS-BI phosphoric acid, 4% acetic acid and 2% tartaric acid. It was left for more than a minute. Observed by light microscopy, TRAP-positive multinucleated cells (TRAP (+) MNCs) with three or more nuclei were counted and used as an index for generating osteoclasts (15).

Experimental Example 3. Effect on Gene Expression

In order to determine the effect on the gene expression of osteoclast differentiation and proliferation-related factors of TRAP, NFATc1, JNK, etc. of the processed ginseng extract of the present invention of Example 1 was performed as follows.

3-1. gun RNA  detach

Total RNA was isolated from TRAP (+) multinuclear cells differentiated into osteoclasts by treatment with 1 ml Trizol solution (Invitrogen, USA). 100 phenol and 100 chloroform: isoamyl alcohol (24: 1) are added to the isolated RNA, and the mixture is mixed well and centrifuged twice to separate the supernatant. RNA is precipitated using 0.5 ml isopropyl alcohol, washed with 70% ethanol and dried naturally. RNA was thawed in RNAase free water (RNA) free water (Promega, USA), followed by the addition of ALNAISE-free DNase (RNase-free DNase, Promega, USA) and stored at -70 ^° C.

3-2. cDNA  Produce

Oligo dT 1 was added to the total RNA solution (containing 13 ug RNA) of each of the control and experimental groups isolated in Experimental Example 3-1, mixed carefully, and then incubated at 70 ^° C. for 5 minutes. Leave primers at room temperature for about 10 minutes to release the primers, then add cyscript buffer, 0.1 M DTT, dUTP nucleotides, dUTP seeded-labeled nucleotides, Cyscript reverse transcriptase, and then add Mix carefully. Thereafter, the cells were incubated at 42 ^° C. for 90 minutes and then left on ice. 2.5 M sodium hydroxide was added thereto, followed by incubation at 37 ^° C. for 15 minutes, and neutralized by adding 2 M HEPES buffer to prepare cDNA.

3-3. Real time RT - PCR

First, 5 ug of RNA, 50 ng / random hexamer (random hexamer) 3, 10 mM dNTP 1, were added to the test tube, and DEPC-H ₂ O was added to make an RNA / primer mixture of 10. The experimental sample was incubated at 65 ° C. for 5 minutes and then left on ice for at least 1 minute. The reaction mixture was prepared by mixing 10-fold RT buffer 2, 25 mM magnesium chloride 4, 0.1 M DTT 2, RNAase (Promega, USA) 1. The reaction mixture was added to the RNA / primer mixture, left to stand at room temperature for 2 minutes, then SuperScript II RT (Promega, USA) 1 (50 units) was added and incubated at 25 ^° C. for 10 minutes. Incubated at 42 ^o C for 50 min, then inactivated by heating at 70 ^o C for 15 min and cooled on ice. RNase (Promega, USA) 1 was added and incubated again at 37 ^o C for 20 min, then stored at -20 ^o C until use. Into each optical tube (optical tube, Gibco, USA) add 2x Cyber Green Mix (SYBR Green Mix, Takara, Japan) 12.5, cDNA 0.2, 5 pmol / primer pair mix 1, 11.3 H ₂ O, 50 ^o Amplification with C 2 min 1 cycle, 95 ^° C 10 min 1 cycle, 95 ^° C 15 sec, 60 ^° C 30 sec, 72 ^° C 30 sec 40 cycles, 72 ^° C 10 min 1 cycle. After completing the PCR, the tube was taken out, and PCR specificity was measured on a 3% agarose gel using 5 ul of the reaction solution. Real time PCR results were analyzed using SDS 7000 software.

When RANKL (receptor activator for nuclear factor B ligand) treatment of RAW264.7 cells increased the expression of TRAP (+) multinucleated cells (MNCs) to promote osteoclast differentiation, the processed ginseng extract of the present invention (SG) Inhibited increased RANKL-induced osteoclast differentiation at concentrations of 0.1 ug / ml, 1 ug / ml (FIG. 1), tartarate resistance Acid Phosphatase (TRAP), JNK, and NFATc1 were RANKL Expression was increased during the treatment, the processed ginseng extract of the present invention (SG) was confirmed to significantly inhibit the gene expression increased in 0.1 ug / ml, 1 ug / ml, concentration (Fig. 2, Fig. 3 and 4). Reference). Therefore, the processed ginseng extract (SG) of the present invention was found to have an excellent effect in inhibiting TRAP, NFATc1, JNK gene expression.

Statistical treatments were compared individually using Student's t-test and determined that there was a significant difference when the p-value was less than 0.05.

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

Formulation example  One. Sanje  Produce

SG Extract 20 mg

Lactose 100 mg

Talc 10 mg

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

Formulation example  2. Preparation of tablets

SG Extract 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

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

Formulation example  3. Preparation of capsules

SG Extract 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed in accordance with a conventional method for producing a capsule, and filled in a gelatin capsule to prepare a capsule.

Formulation example  4. Preparation of injections

SG Extract 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation example  5. Liquid  Produce

SG Extract 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

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

Claims (5)

Bone metabolic disease containing as an active ingredient a processed Panax genus plant extract with an increased active ingredient such that the sum of the ginsenosides Rg3, Rk1, and Rg5 is higher than the sum of the ginsenosides Rb1, Rb2, Rc, and Rd. Pharmaceutical compositions for the treatment and prophylaxis. The method of claim 1,
The Panax genus plants Ginseng (Panax ginseng ), Panax ginseng ( Panax quinquefolia ), Whole Chisam (Samchi, Panax notoginseng ), Vietnamese Ginseng ( Panax vietnamensis ), Panax ginseng ( Panax japonicus ), Panax elegatior , Panax wanzianus ( Panax wangianus , Panax bipinnatifidus ), or Panax pseudoginseng ) is artificially processed. The method of claim 1,
The Panax genus plants are treated with ginseng at a high temperature of 120 to 180 ° C. for 0.5 to 20 hours to provide a medicinal ingredient such that the ratio of ginsenosides (Rg 3 + Rg 5) / (Rc + Rd + Rb ₁ + Rb 2) is 1.0 or more. Pharmaceutical composition characterized by increased processed ginseng. The method of claim 1,
Wherein the extract is water, a lower alcohol such as methanol, ethanol, butanol, or a mixed solvent thereof. The pharmaceutical composition of claim 1, wherein the bone metabolic disease is osteoprosis, paget disease, periodontal disease, metastatic cancer, or rheumatoid arthiritis.

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