KR20130059559A - Pharmaceutical composition comprising extract of amyda sinensis showing an anti-osteoporosis activity - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing an Amyda sinensis extract is provided to effectively suppress osteoclast activity causing bone diseases, and to treat senile bone diseases or bone diseases caused by menopause. CONSTITUTION: A pharmaceutical composition for preventing and treating bone metabolism contains an Amyda sinensis extract as an active ingredient, which suppresses osteoporosis. The metabolic bone diseases are osteoporosis, Paget's disease, periodontal disease, metastatic cancer, or rheumatoid arthritis. The extract contains 0.01-99wt% of a crude drug extract.

Description

Pharmaceutical composition comprising extract of Amyda sinensis showing an anti-osteoporosis activity

The present invention relates to a pharmaceutical composition comprising the extract of Tortoisepa showing an anti-osteoporosis activity 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. Professor of the National College of Oriental Medicine. Herbology. Book publishing company; 603-604: 1994

11. 趙美貞. Enzymatic Hydrolysis and Their Antioxidant Activity of the Extract of Tortoiseshell Extract.全 南大 學校 校 大 學院, 2008

Bone 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). Most of them show osteoporosis by inhibiting osteoblast function or increasing osteoclast activity. Therefore, inhibiting osteoclast function can treat or prevent osteoporosis.

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, MITF, Src, Akt activation, etc. Induces bone resorption by promoting the expression of osteoclast-specific proteins such as cathepsin K and calcitonin receptor (7-9).

Amyda sinensis has a mild temper, and has the effect of 補 肝腎, 滋陰 潛 陽,. In oriental medicine, 陰虛 發熱, 骨蒸 勞 熱, 虛 風 內 動, 經 閉,, 久It was used to treat the back (10). In addition, recent studies have reported that Tortoiseshell has an antioxidant effect (11).

Accordingly, the present inventors have completed the present invention by confirming the osteoporosis inhibitory effect by inhibiting the gene expression of cathepsin K, TNFa, IL-6, etc. Was done.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment and prevention of bone metabolic diseases, which contains as an active ingredient extract of sesame persimmon exhibiting osteoclast inhibitory effect.

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

Below is the extract of the present invention is extractable from the tortoiseshell.

For example, the extract of the present invention, after drying the tortoiseshell, is about 1 to 100 times the sample weight, preferably about 1 to 50 times (w / v) volume of water, C ₁ to C ₄ lower Alcohol or a mixed solvent thereof, preferably water or water and ethanol as an extraction solvent, about 2 to 7 days at a reaction temperature of about 10 to 120, preferably 30 to 90, preferably 12 hours to A second step of repeated extraction 1 to 10 times, preferably 1 to 5 times by a conventional extraction method such as heat extraction, ultrasonic extraction, reflux extraction, ultrahigh pressure extraction for 26 hours, preferably reflux extraction; A third step of filtering and extracting the extract obtained in the above step under reduced pressure; Through the step comprising the manufacturing method of the fourth step of freeze-drying the concentrated extract can be obtained the extract of the present invention.

In another aspect, the present invention provides a pharmaceutical composition for the treatment and prevention of bone metabolic diseases containing the preparation method and tortoiseshell extract prepared by the production method.

Asparagus extract according to the present invention shows the inhibitory effect of osteoclast differentiation, osteoclast differentiation and proliferation-related factors, such as cathepsin K, TNFa, IL-6, gene expression inhibitory effect, it was confirmed that it is useful as a pharmaceutical composition for inhibiting osteoporosis.

The extract of the present invention comprises 0.01 to 99% by weight of the herbal extract based on the total weight of the extract.

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.

The composition comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly 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 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 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 extract of the present invention can be administered to mammals such as rats, mice, livestock, humans and the like in various routes. All modes of administration can be anticipated, for example, by oral and rectal or intravenous methods.

Asparagus extract according to the present invention exhibits the inhibitory effect of osteoclast differentiation (TRAP (+) multinucleated cell generation), the gene expression inhibitory effect of cathepsin K, TNFa, IL-6, etc. It can be usefully used as an inhibitory pharmaceutical composition.

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 Tortoiseshell extract (AS),
Figure 2 is a diagram showing the effect on the expression of Cathepsin K according to the concentration of Tortoiseshell extract (AS),
Figure 3 is a diagram showing the effect on the expression of TNFa according to the concentration of Tortoiseshell extract (AS),
Figure 4 is a diagram showing the effect on IL-6 expression according to the concentration of Tortoiseshell extract (AS).

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

Example 1. Preparation of Tortoisenium Extract

After adding 10% of 30% ethanol to the tortoiseshell, hot water was extracted for 4 hours, and the extract was concentrated and then freeze-dried and used as a sample of the following experimental example (hereinafter, AS).

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 (1) RANKL-treated control group (N) (2) RANKL-induced control group (C), (3) RANKL-induced group + group administered the 10ug / ml tortoiseshell extract of Example 1 (AS50), (4) RANKL-induced group + group administered the 100ug / ml Tortoiseshell extract of Example 1 (AS100), (5) RANKL-induced group + group administered the 200ug / ml Tortoiseshell extract of Example 1 (AS200).

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 (9).

Experimental Example  3. Impact on Gene Expression

In order to determine the effect on the gene expression of the osteoclast differentiation and proliferation-related factors, such as Cathepsin K, TNFa, IL-6 of the extract of Example 1 was carried out 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.

Treatment of receptor activator for nuclear factor B ligand (RANKL) in RAW264.7 cells increased the expression of TRAP (+) multinucleated cells (MNCs) to promote osteoclast differentiation. Inhibitors of receptor activator for nuclear factor B ligand (RANKL) -induced osteoclast differentiation were observed at ug / ml and 200 ug / ml concentrations (FIG. 1). , It was confirmed that the extract of the Tortoiseshell significantly inhibited the increased gene expression at a concentration of 50 ug / ml, 100 ug / ml, and 200 ug / ml (see FIGS. 2, 3, and 4). Therefore, Tortoiseshell extract was found to have an excellent effect on the inhibition of Cathepsin K, TNFa, IL-6 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

Tortoiseshell Extract 200 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

Tortoiseshell Extract 200 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

Tortoiseshell Extract 200 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

Tortoiseshell Extract 200 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation example  5. Liquid  Produce

Tortoiseshell Extract 200 mg

10 g per isomer

5 g mannitol

Purified water

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 (2)

Pharmaceutical composition for the treatment and prevention of bone metabolic diseases, containing as a active ingredient extract of Tortoiseshell extract showing inhibitory effect on osteoporosis. The method of claim 1,
The bone metabolic disease is osteopharmaceuticals (osteoprosis), Paget disease (paget disease), periodontal disease (periodontal disease), metastatic cancer (rheumatoid arthiritis) pharmaceutical composition (rheumatoid arthiritis).

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