KR20130049560A - Health functional food comprising poncirin showing an inhibition effect of osteoclastogenesis - Google Patents

Abstract

PURPOSE: Provided is a health functional food for preventing and controlling bone and joint diseases, the food containing poncirin as an active ingredient, wherein poncirin has an osteoclast differentiation inhibiting effect. CONSTITUTION: A health functional food contains poncirin having an osteoclast differentiation inhibiting effect. The health functional food is effective in preventing and controlling a metabolic bone disease. The metabolic bone disease is osteoporosis, Paget's disease, periodontal disease, metastatic cancer, or rheumatoid arthritis.

Description

Health functional food comprising Poncirin showing an inhibition effect of osteoclastogenesis}

The present invention relates to a health functional food having an osteoclast activity inhibitory effect containing ponsyrin 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. Zheng H, Yu X, Collin-Osdoby P, Osdoby P. RANKL stimulates inducible nitric-oxide synthase expression and nitric oxide production in developing osteoclasts. An autocrine negative feedback mechanism triggered by RANKL-induced interferon-beta via NF-kappaB that restrains osteoclastogenesis and bone resorption. J Biol Chem. 2006; 281 (23): 15809-20.

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. Liu XH, Kirschenbaum A, Yao S, Levine AC. Interactive effect of interleukin-6 and prostaglandin E2 on osteoclastogenesis via the OPG / RANKL / RANK system. Ann N Y Acad Sci. 2006; 1068: 225-33.

10. Rajkumar S, Jebanesan A. Bioactivity of flavonoid compounds from Poncirus trifoliata L. (Family: Rutaceae) against the dengue vector, Aedes aegypti L. (Diptera: Culicidae). Parasitol Res. 2008; 104 (1): 19-25

11.Kim JB, Han AR, Park EY, Kim JY, Cho W, Lee J, Seo EK, Lee KT. nhibition of LPS-induced iNOS, COX-2 and cytokines expression by poncirin through the NF-kappaB inactivation in RAW 264.7 macrophage cells. Biol Pharm Bull. 2007; 30 (12): 2345-51.

12.Lee JH, Lee SH, Kim YS, Jeong CS. Protective effects of neohesperidin and poncirin isolated from the fruits of Poncirus trifoliata on potential gastric disease. Phytother Res. 2009; 23 (12): 1748-53.

13.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.

14. Korean patent registration number (date) 1008247040000 (20080417)

15. Korea Patent Registration Number (Date) 1003228760000 (20020118)

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).

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 catarsin TRAP (tartarate resistant acid phosphatase) through activation of NF-B, c-Fos, c-jun, AP-1, NFATc1, MAPK, ERK, JNK, p38 activation process, Src, Akt activation It promotes the expression of osteoclast specific proteins such as K and calcitonin receptor (7-9).

Poncirin is known as the main component of the chamber (10), and pharmacologically reported anti-inflammatory effects (11) and anti-gastric ulcer effects (12).

Accordingly, the present inventors have completed the present invention by confirming the effect of inhibiting osteoclast differentiation of poncirin, gene expression inhibitory factors such as TRAP, Cathepsin K, MMP-9 expression, NFATc1, etc. It became.

In order to achieve the above object, the present invention provides a functional food for preventing and improving osteoarthritis disease containing as a active ingredient ponsirin (Poncirin) showing the 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.

The compounds of the present invention are commercially available but can be separated from plant extracts as follows.

For example, the compounds and extracts of the present invention, after washing and drying the fruiting chamber, have a volume of water of about 1 to 100 times the sample weight, preferably about 1 to 50 times (w / v), C ₁ to Lower alcohol of C ₄ 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 The second step of extracting 1 to 10 times, preferably 1 to 5 times by a conventional extraction method, such as heating extraction, ultrasonic extraction, reflux extraction, ultra-high pressure extraction method for 12 to 26 hours, preferably reflux extraction; A third step of filtering and extracting the extract obtained in the above step under reduced pressure; The fruit extract of the present invention is obtained through a step including a method of preparing the freeze-dried extract of the concentrated extract, and the purification process such as silica gel column chromatography or Sephadex column chromatography is repeated several times. Compounds of

In another aspect, the present invention provides a pharmaceutical composition, health functional food, or food additives for the treatment and prevention of bone metabolic diseases containing the preparation method and the ponsyrin prepared by the production method as an active ingredient.

Poncirine according to the present invention shows an inhibitory effect on osteoclast differentiation, osteoclast differentiation and proliferation-related factors such as TRAP, Cathepsin K, MMP-9, NFATc1, etc. It has been found to be useful as a food or food additive.

The compounds and extracts of the present invention comprise 0.01 to 99% by weight of the herbal extracts relative to the total weight of the compounds and extracts.

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 compounds and extracts of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Compositions comprising compounds and extracts according to the invention are in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents that may be included in the formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, 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 compound and extract is preferably administered at 0.01 mg / kg to 10 g / kg, 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 compounds and extracts of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral and rectal or intravenous methods.

In another aspect, the present invention provides a health functional food for the improvement and prevention of bone metabolic diseases containing ponsyrin as an active ingredient.

Health functional foods containing phonicrin of the present invention as an active ingredient can be used in various ways, such as drugs, foods and beverages for the prevention and improvement of bone metabolic diseases. Foods to which the compounds and extracts of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, leach teas, health supplements, and the like, and are powders, granules, tablets, capsules or beverages. Available in form.

Therefore, the present invention also provides a food or food additive containing ponsyrin as an active ingredient having an effect of preventing and improving bone metabolic diseases.

Food forms to which the compounds and extracts of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, or health supplements of candy.

The compounds and extracts of the present invention may be added to foods or beverages for the purpose of preventing and ameliorating bone metabolic diseases. At this time, the amount of the compound and extract in the food or beverage is generally added to the health food composition of the present invention 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 10 g based on 100 ml, Preferably it can be added in the ratio of 0.3-1 g.

The health beverage composition of the present invention contains a mixture of the compounds and extracts as essential ingredients in the ratios indicated, and there are no particular limitations on the liquid ingredients, and it contains various flavors or natural carbohydrates as additional ingredients, like ordinary drinks. can do. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the compounds and extracts of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the compounds and extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Poncirine according to the present invention shows an inhibitory effect on osteoclast differentiation, osteoclast differentiation and proliferation-related factors such as TRAP, Cathepsin K, MMP-9, NFATc1, etc. Food, it can be usefully used as a food additive.

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 poncirin (Poncirin),
2 is a diagram showing the effect on TRAP expression according to the concentration of ponsirin (Poncirin),
3 is a diagram showing the effect on the expression of Cthepsin K according to the concentration of ponsirin (Poncirin),
4 is a diagram showing the effect on the expression of MMP-9 according to the concentration of ponsirin (Poncirin),
5 is a diagram showing the structure of poncirin (poncirin).

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 Fonsirine

It was used as a sample of the following experimental example after being sold by ponsulin (Food and Drug Administration).

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 (1) RANKL-treated control group (C) (2) RANKL-induced control group (C), (2) RANKL-induced group + 1 / group administered the ponsirin (Poncirin) of Example 1 (PCN1), (3) RANKL induction group + 5 / It was set as the group (PCN5) to which the poncirin 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) having three or more nuclei were counted and used as an index for generating osteoclasts (13).

Experimental Example 3. Effect on Gene Expression

Experiments were performed as follows to determine the effects on gene expression such as TRAP, Cathepsin K, MMP-9 expression inhibition, NFATc1, etc., which are factors related to osteoclast differentiation and proliferation of ponsyrin. .

3-1. gun RNA  detach

Cultures of B16F10 cells were treated with 1 ml Trizol solution (Invitrogen, USA) to isolate total RNA. 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 RNAase-free DNase (RNase-free DNase, Promega, USA) and stored at -70.

3-2. cDNA  Produce

OligodT 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 expression of TRAP (+) multinucleated cells (MNCs) to promote osteoclast differentiation. ug / ml, it was shown to inhibit the receptor activator for nuclear factor B ligand (RANKL) -induced osteoclast differentiation at concentrations (FIG. 1) (tartarate resistance Acid Phosphatase), Cathepsin K, MMP-9, NFATc1 when RANKL treatment Expression was increased, and the phonicrin was found to significantly inhibit the gene expression increased at 1 ug / ml, 5 ug / ml, concentration (see Fig. 2, 3 and 4). Thus, it can be seen that ponsyrin has an excellent effect on inhibiting tyrosinase, TRAP, Cathepsin K, MMP-9, and NFATc1 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 compound 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

Ponsiline 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

Ponsiline 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

Ponsiline 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

Ponsiline 200 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

Ponsiline 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.

Formulation example  6. Manufacture of health food

Ponsyrine 1000 mg

Vitamin mixture quantity

Vitamin A Acetate 70

Vitamin E 1.0

Vitamin B1 0.13

Vitamin B2 0.15

Vitamin B6 0.5

Vitamin B12 0.2

Vitamin C 10

Biotin 10

Nicotinamide 1.7

Folic acid 50

Calcium Pantothenate 0.5

Mineral mixture quantity

Ferrous Sulfate 1.75

Zinc Oxide 0.82

Magnesium Carbonate 25.3

Potassium Phosphate Monobasic 15

Dicalcium Phosphate 55

Potassium Citrate 90

Calcium Carbonate 100

Magnesium chloride 24.8

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

Ponsyrine 1000 mg

Citric Acid 1000

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water is added to the total 900

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 for about 1 hour. The resulting solution was filtered and sterilized in a sterilized 2 l vessel. The resulting solution was refrigerated, Used in the manufacture of health beverage compositions.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (2)

Health functional food for the prevention and improvement of bone joint disease containing ponsulin (Poncirin) showing the osteoclast inhibitory effect as an active ingredient.
The method of claim 1,
The bone metabolic disease is osteoporosis (osteoprosis), Paget disease (paget disease), periodontal disease (periodontal disease), metastatic cancer (rheumatoid arthiritis) health functional food.

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