KR20130060586A - Health functional food comprising extract of cordyceps militaris showing an inhibitory effect of osteoclast differentiation - Google Patents
Health functional food comprising extract of cordyceps militaris showing an inhibitory effect of osteoclast differentiation Download PDFInfo
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- KR20130060586A KR20130060586A KR1020110126721A KR20110126721A KR20130060586A KR 20130060586 A KR20130060586 A KR 20130060586A KR 1020110126721 A KR1020110126721 A KR 1020110126721A KR 20110126721 A KR20110126721 A KR 20110126721A KR 20130060586 A KR20130060586 A KR 20130060586A
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- extract
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- osteoclast
- osteoclast differentiation
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A23V2200/00—Function of food ingredients
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- A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The present invention relates to a health functional food of Militaris cordyceps sinensis extract that inhibits osteoclast differentiation, and to imbalance due to an increase in bone resorption for bone formation in bone remodeling. The present invention relates to a health functional food that can effectively suppress the increase in osteoclast activity, which is a major cause of bone diseases, and thus help to improve bone diseases caused by bone diseases of the elderly or menopause of women. More specifically, the health functional foods including osteoporosis, including osteoporosis, by inhibiting osteoclast differentiation and inhibiting osteoclast activity including osteoclast inhibition of TRAP (tartarate resistance Acid Phosphatase), inhibition of JNK expression, and inhibition of AKT1 expression. It is about.
Description
The present invention relates to a health functional food containing Militaris cordyceps sinensis extract that inhibits osteoclast differentiation as an active ingredient.
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Mizuha Y, Yamamoto H, Sato T, Tsuji M, Masuda M, Uchida M, Sakai K, Taketani Y, Yasutomo K, Sasaki H, Takeda E. Water extract of Cordyceps sinensis (WECS) inhibits the RANKL-induced osteoclast differentiation . Biofactors. 2007; 30 (2): 105-16.
15. 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.
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 in women, hyperthyroidism, diabetes, stress, smoking and lack of exercise, physical aging and the use of glucocorticoid drugs (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; Reddy SV.Regulatory mechanisms operative in osteoclasts.Crit Rev Eukaryot Gene Expr. 2004, 14 (4): 255-70; Pang M, Martinez AF, Fernandez I, Balkan W, Troen BR.AP-1 stimulates the cathepsin K promoter in RAW 264.7 cells.Gen., 2007, 403 (1-2): 151-8).
Osteoclasts are differentiated by various differentiation-causing factors in macrophage progenitor cells (Boyce BF, Yamashita T, Yao Z, Zhang Q, Li F, Xing L. Roles for NF-kappaB 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). 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. Induce differentiation and activation (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). 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 Promote osteoclast specific protein expression such as K, calcitonin receptor (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; 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-kap paB signaling pathways.Eur J Pharmacol. 2010, 636 (1-3): 28-35).
Militaris cordyceps (Cordyceps militaris) is a small mushroom that drops fruiting bodies to insect bodies, some of which are known as host. It is mainly used for symptoms of low back and knee pain, lower body weakness, oil well, dreaming, premature ejaculation, tinnitus, forgetfulness, mental ecstasy, flatulence, seawater, and cold sweating after chronic illnesses. Mycelial growth of Cordyceps militaris, 2002. 30 (1): 1-5). The main component of Militaris cordyceps is known as cordycepin, which contains about 2% in Cordyceps militaris, and anticancer activity (Park SE, Kim J, Lee YW, Yoo HS, Cho CK.Antumor activity of water extracts from Cordyceps militaris in NCI-H460 cell xenografted nude mice.J Acupunct Meridian Stud. 2009; 2 (4): 294-300), Lee JS, Hong EK.Immunostimulating activity of the polysaccharides isolated from Cordyceps militaris.Int Immunopharmacol. 2011 11 (9): 1226-33), Oh JY, Choi WS, Lee CH, Park HJ.The ethyl acetate extract of Cordyceps militaris inhibits IgE-mediated allergic responses in mast cells and passive cutaneous anaphylaxis reaction in mice J Ethnopharmacol. 2011; 135 (2): 422-9). Osteoclast differentiation efficacy of Cordyceps sinensis, another species of Cordyceps sinensis, has been reported (Mizuha Y, Yamamoto H, Sato T, Tsuji M, Masuda M, Uchida M, Sakai K, Taketani Y, Yasutomo K, Sasaki H, Takeda E. Water extract of Cordyceps sinensis (WECS) inhibits the RANKL-induced osteoclast differentiation.Biofactors. 2007; 30 (2): 105-16), the effects on osteoblasts and osteoclasts and osteoporosis effects on Cordyceps militaris Not reported.
Accordingly, the present inventors have completed the present invention by confirming the effect of inhibiting the osteoclast differentiation, osteoclast differentiation and osteoclast activity related factors such as TRAP, JNK, AKT1, etc., of the extract of Cordyceps militaris. .
In order to achieve the above object, the present invention provides a dietary supplement for preventing and improving bone metabolic disease, which contains as an active ingredient Millitaris Cordyceps sinensis extract showing an osteoclast inhibitory effect.
The militaris Cordyceps sinensis extract of the present invention is commercially available but can be extracted from the millitaris cordyceps sec.
For example, the extract of the present invention, after drying the millitaris Cordyceps sinensis, 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 a mixed solvent of water and ethanol, and a mixed solvent of 10 to 60% ethanol, about 10 to 120, preferably about 1 hour at a reaction temperature of 30 to 90 1 to 10 days, preferably 2 to 15 hours, such as heating extraction, ultrasonic extraction, reflux extraction, ultra-high pressure extraction, such as conventional extraction methods, preferably reflux extraction 1 to 10 times, preferably 1 to 5 times Extracting a second step; A third step of filtering and extracting the extract obtained in the above step under reduced pressure; Millitaris Cordyceps sinensis extract of the present invention can be obtained through the step comprising the method of the fourth step of freeze-drying the concentrated extract.
In another aspect, the present invention provides a health functional food, or a food additive for the prevention and improvement of bone metabolic diseases containing the method and the millitaris Cordyceps sinensis extract prepared by the method.
The bone metabolic disease as defined herein includes osteoprosis, paget disease, periodontal disease, metastatic cancer or rheumatoid arthiritis, preferably osteoporosis. Characterized in that.
Militaris Cordyceps sinensis extract according to the present invention exhibits inhibitory effects on osteoclast differentiation, osteoclast differentiation and proliferation related gene expression, such as TRAP, JNK, AKT1, and has been found to be useful as a health functional food 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 as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent 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.
In another aspect, the present invention provides a health functional food containing Militaris Cordyceps sinensis extract as an active ingredient for the prevention and improvement of bone metabolic diseases.
Health functional foods containing the extract Millitaris cordyceps sinensis of the present invention as an active ingredient may be used in various ways, such as drugs, foods and beverages for the prevention and improvement of bone metabolic diseases. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamin complex, leach tea, health supplement foods and the like, and they are in the form of powder, granule, tablet, capsule or beverage Can be used.
Therefore, the present invention also provides a food or food additive containing millilis cordyceps sinensis extract as an active ingredient having an effect of preventing and improving bone metabolic diseases.
The food forms to which the extract of the present invention can be added include various foods of candy, beverages, gums, tea, vitamin complexes, or foods that are health supplement foods.
Extracts of the present invention may be added to food or beverages for the purpose of preventing and improving bone metabolic diseases. At this time, the amount of the extract in the food or beverage is generally added to the health food composition of the present invention to 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 Can be added at a ratio of 0.3 to 1 g.
The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient, such as ordinary beverages, in addition to containing a mixture of the above extract as an essential ingredient in the indicated ratios, have. 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 extract of the present invention includes 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 its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components may 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.
Militaris cordyceps sinensis extract according to the present invention exhibits the inhibitory effect of osteoclast differentiation, osteoclast differentiation and proliferation-related factors, such as TRAP, JNK, AKT1, etc. Can be.
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 Militaris Cordyceps sinensis extract,
Figure 2 is a diagram showing the effect on TRAP expression according to the concentration of Militaris Cordyceps Sinensis,
Figure 3 is a diagram showing the effect on the expression of JNK according to the concentration of Militaris Cordyceps Sinensis,
Figure 4 is a diagram showing the effect on AKT1 expression according to the concentration of Militaris Cordyceps Sinensis.
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 Millitaris Cordyceps Sinensis Extract
Militaris Cordyceps sinensis was added to 30
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 / of Millitaris Cordyceps Sinensis extract of Example 1 (CM 0.1) , (3) RANKL induction group + 5 / to the group to which the milliliris cordyceps extract of Example 1 was administered (CM1).
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 as markers for the generation of osteoclasts (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 ).
Experimental Example 3. Effect on Gene Expression
Experiments were performed as follows to determine the effect on gene expression of factors related to osteoclast differentiation and proliferation of the Millitaris cordyceps sinensis extract of Example 1, such as TRAP, JNK, AKT1.
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
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, and in this example, the extract of Militaris cordyceps was 0.1 ug /. ml, 1 ug / ml, it was shown to inhibit the receptor activator for nuclear factor B ligand (RANKL) -induced osteoclast differentiation at concentrations (FIG. 1) .TAP (tartarate resistance Acid Phosphatase), JNK, and AKT1 were expressed during RANKL treatment. It was confirmed that the Millitaris cordyceps sinensis extract significantly inhibited the gene expression increased at 0.1 ug / ml, 1 ug / ml (see FIGS. 2, 3 and 4). Therefore, Militaris cordyceps sinensis extract was found to have an excellent effect in inhibiting TRAP, JNK, AKT1 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.
Preparation Example 1. Preparation of powder
Militaris Cordyceps Sinensis Extract 200 mg
Lactose 100 mg
The above components are mixed and filled in airtight bags to prepare powders.
Formulation Example 2. Preparation of tablets
Militaris Cordyceps Sinensis Extract 200 mg
Corn starch 100 mg
Lactose 100 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
Militaris Cordyceps Sinensis Extract 200 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. Liquid Produce
Militaris Cordyceps Sinensis 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 (3)
The dietary supplement is a powder, tablets, calcels, liquid dietary supplements.
The bone metabolic diseases include osteoporosis (osteoprosis), Paget disease (paget disease), periodontal disease (periodontal disease), metastatic cancer (metastatic cancer) or rheumatoid arthritis (rheumatoid arthiritis) characterized in that it comprises.
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| WO2020241958A1 (en) * | 2019-05-28 | 2020-12-03 | 동아제약 주식회사 | Composition for preventing or treating woman menopause symptoms, comprising cordyceps militaris concentrate as active ingredient |
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