WO2013176327A1 - Pharmaceutical composition and health functional food for preventing or treating bone diseases, containing lactic acid bacteria fermentation product of detox soup of coptis chinensis as active ingredients - Google Patents

Description

Pharmaceutical composition and health functional food for the prevention or treatment of bone disease containing lactic acid bacteria fermentation product of Hwangnyeonhaedoktang as an active ingredient

The present invention relates to a pharmaceutical composition for preventing or treating bone diseases and a health functional food containing the lactic acid bacteria fermentation product of Hwangnyeonhaedoktang as an active ingredient.

Normal bone remodeling is a balance between bone formation and bone resorption, and bone formation and bone resorption are largely caused by the interaction of three cells: chondrocytes, osteoblasts and osteoclasts. . In particular, osteoblast proliferation and differentiation are closely related to bone calcification. Bone calcification does not occur when RUNX2, which is an important transcription factor in osteoblasts, decreases or the expression level decreases. RANKL (receptor activator of NF-kB ligand) is expressed during osteoblast differentiation and is known as a factor promoting osteoclast differentiation. OPG (Osteoprotegrin) is also expressed in osteoblasts to inhibit the role of RANKL to differentiate osteoclasts It is known as a factor that suppresses.

Bones support the soft tissues and weight of the body and surround internal organs to protect internal organs from external shocks. It is also an important part of the body that not only structurally supports muscles or organs, but also stores calcium and other essential minerals in the body, such as phosphorus and magnesium. Thus, the bones of grown adults do not stop, and the balance of the generation and absorption process that removes old bones and replaces them with new bones until the day of death is very dynamic and continuous. This is called bone remodeling. Bone circulation, which removes old bone and replaces it with new bone, is essential to repair the microscopic damage caused by growth and stress and to maintain proper bone function.

It is known that two types of cells are involved in aggregate formation. One of the two cells is osteoblasts that produce bone, and the other is osteoclasts that destroy bone. Osteoblasts produce RANKL and its inducing receptor, OPG. When RANKL binds to RANK, a receptor on the surface of osteoclast progenitor cells, osteoclast progenitor cells mature into osteoclasts, resulting in bone resorption. However, when OPG binds to RANKL, the binding between RANKL and RANK is blocked, so that the formation of osteoclasts is suppressed and bone resorption does not occur more than necessary.

Osteoporosis is a condition in which bone mass decreases due to various causes and the risk of fracture is continuously increased due to the deterioration of the microstructure of bone tissue. Osteoporosis is a condition in which minerals (particularly calcium) and substrates that make up bone are reduced. Formation is broken and osteoclasts occur in an increased state than osteopathy. Normal bone inside has a dense structure like a net, but in the case of osteoporosis, the gap between bone microstructures becomes thinner and the microstructure becomes thinner and weaker, which increases the risk of bone fracture even after a small impact. It is classified as elderly osteoporosis, which occurs gradually in men and women over 70 years of age and causes progressive bone loss of the pelvis and vertebra, and secondary osteoporosis due to diseases, drugs, alcohol, smoking, or accidents, regardless of age.

Osteoporosis is currently one of the most important social problems in the United States, causing about 260,000 women every year, of which about 12 to 20% of deaths are caused. Aging and postmenopausal women's osteoporosis and osteoporosis fractures cause serious problems in an aging society and women's social participation.

Materials currently used for the treatment of osteoporosis include estrogen, androtic anagolic ateroids, calcium preparations, ginseng salts, fluoride preparations, ipriflavones, vitamin D ₃ and the like. Estrogens inhibit osteoblastic cell death, increase cell survival, and promote osteoclast cell death to decrease cell survival, which is somewhat effective in treating menopausal symptoms and maintaining bone mineral density. There are side effects that cause back. In addition, there are calcitonin, parathyroid hormone, bisphosphonate preparations, etc. as drugs that inhibit the activity of osteoclasts to inhibit bone destruction or increase the activity of bone regeneration units through the proliferation of osteoblasts. However, existing osteoporosis therapeutic agents have only the effect of blocking bone resorption or promoting bone formation and causing many side effects upon long-term administration. Therefore, there is a need for the development of safe prophylactic and therapeutic agents that have a long-term effect of increasing bone mineral density and have fewer side effects.

Therefore, while the present inventors are studying natural medicines for preventing or treating bone diseases such as osteoporosis as described above, lactic acid bacteria fermented products of mixed herbal extracts of rhubarb, gold, yellow white and gardenia increase the bone density of ovarian isolated animals, TRAP By showing the activity and inhibition of the formation of multinucleated osteoclasts, the present invention was confirmed to be effective in the prevention or treatment of bone diseases.

An object of the present invention is to provide a pharmaceutical composition or health functional food containing a lactic acid bacteria fermentation product of Hwangnyeonhaedoktang as an active ingredient that can prevent or treat bone diseases.

In order to solve the above problems, it provides a pharmaceutical composition for the prevention or treatment of bone diseases comprising the lactic acid bacteria fermentation product of Hwangnyeonhaedoktang as an active ingredient.

The term 'hwangryeonhaedoktang' used in the present invention is composed of sulfur, golden, yellow, white and gardenia, and herbal medicine that is effective when there is a fever of three seconds (meaning, upper, middle, and lower) Means derivation. It is known to be prescribed when there is inflammation due to febrile disease, mental instability due to fever and hyperemia, redness of the yoga and deep subsidence, and also for prescription bleeding, hemoptysis, and cerebral hemorrhage. Recent studies have reported antioxidant, anti-ulcer, diuretic, hypotensive and antimicrobial effects, and have been reported to have effects on atopic dermatitis, contact dermatitis and seborrheic dermatitis in relation to skin. . Hwangnyeonhaedoktang of the present invention is preferably a hot water extract of sulfur, golden, yellowish white and gardenia.

The term 'lactic acid bacteria fermented product' used in the present invention means a culture result obtained from a culture inoculated with lactic acid bacteria and cultured on a medium in which lactic acid bacteria can grow. The lactic acid bacteria fermentation product of the present invention is inoculated with lactic acid bacteria to the Hwangnyeonhaedoktang, fermented under an aerobic atmosphere for a temperature of 35 ℃ to 40 ℃ and 1 to 3 days.

The lactic acid bacteria is preferably Lactobacillus (Lactobacillus) or Bifidobacterium (Bifidobacterium) genus.

The genus Lactobacillus (Lactobacillus), Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus plantarum, Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus fermentum At least one selected from the group consisting of Lactobacillus curvatus, Lactobacillus confusus and Lactobacillus gasseri strains is preferred.

The Bifidobacterium genus is preferably a Bifidobacterium breve or Bifidobacterium thermophilum strain.

The term 'bone disease' used in the present invention refers to all diseases caused by a decrease in bone density, and includes osteoporosis, osteoplasia, periodontal disease or fracture.

In the present invention, it was confirmed that the composition containing the lactic acid bacteria fermentation product of Hwangnyeonhaedoktang is effective in the prevention or treatment of bone diseases. According to an embodiment of the present invention, by identifying the osteoclast differentiation marker TRAP activity and multinuclear osteoclast formation inhibitory effect that is an indicator of bone disease, it was confirmed that there is an effect of preventing or treating bone disease.

As used herein, the term 'prevention' means all actions of inhibiting or delaying a disease by administration of a composition containing the lactic acid bacterium fermentation product of the Hwangnyeonhaedoktang. In addition, the term 'treatment' used in the present invention means all the actions that improve or cure the symptoms of the disease by administration of the composition containing the lactic acid bacteria fermentation product of the Hwangnyeonhaedoktang.

The composition of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described active ingredient for administration. The carrier, prosthetic agent and diluent 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, talc, magnesium stearate and mineral oil.

The compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, or the like, oral preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. Specifically, when formulated, it may be prepared by using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrating agents, and surfactants which are commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like in the lactic acid bacterium fermentation product of Hwangnyeonhaedoktang. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. It may be prepared by adding various excipients such as humectants, sweeteners, fragrances, preservatives and the like in addition to liquid oral liquids or liquid paraffin for oral use. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and tasks. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrosol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the condition and weight of the patient, the extent of the disease, Depending on the drug form, route of administration, and time, it may be appropriately selected by those skilled in the art. The daily dosage of the lactic acid bacterium fermentation product of the Hwangnyeonhaedoktang is preferably 1 mg / kg to 500 mg / kg, and may be administered once to several times daily if necessary.

The present invention also provides a health functional food composition for the prevention or improvement of bone diseases containing the lactic acid bacteria fermentation product of Hwangnyeonhaedoktang as an active ingredient.

The health functional food 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 salts thereof, Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. It may also contain natural fruit juices and pulp for the production of fruit juices and vegetable drinks. These components can be used independently or in combination. The dietary supplement may be in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol and vitamin complex. Can be.

In addition, the health functional food may further include food additives, and the suitability as a "food additive" is related to the relevant items according to the General Regulations and General Test Act of the Food Additives Code approved by the Food and Drug Administration unless otherwise specified. Judging by the standards and standards.

Items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, cinnamon acid, natural additives such as color pigments, licorice extract, crystalline cellulose, high-quench pigments, guar gum, L Mixed preparations, such as a sodium glutamate preparation, a noodles addition alkali preparation, a preservative preparation, and a tar pigment preparation, etc. are mentioned.

At this time, the lactic acid bacteria fermented product of Hwangnyeonhaedoktang according to the present invention, which is added to foods including beverages in the process of manufacturing health functional foods, may be appropriately added or subtracted thereto, preferably 1 to 100% by weight of food. It is preferable to add to 15 to 15% by weight.

The present invention is a lactic acid bacteria fermentation product of Hwangnyeonhaedoktang effective in the prevention or treatment of bone diseases, as well as pharmacologically available as a preventive or therapeutic composition for bone diseases can be useful as a health functional food.

1 is a graph showing the effect on TRAP activity of Hwangnyeonhaedoktang (HRT) and Hwangnyeonhaedoktang fermented product (fHRT) in RAW264.7 cells according to an embodiment of the present invention.

Figure 2, (a) TRAP activity inhibitory effect, (b) TRAP-positive multinucleated osteoclast formation inhibitory effect of Hwangnyeonhaedoktang (HRT) and Hwangnyeonhaedoktang fermented product (fHRT) according to an embodiment of the present invention ( c) The results of the cell photos showing the inhibitory effect of TRAP-positive multinucleated osteoclast formation were shown.

Figure 3, (a) ethyl acetate (EA) fraction of TRAP activity inhibitory effect of Hwangnyeonhaedaktang (HRT) and Hwangnyeonhaedoktang fermented product (fHRT) according to an embodiment of the present invention, (b) butanol (BU) The results of the cell photos showing the inhibitory effect of the fraction of TRAP activity, (c) butanol (BU) fraction of TRAP positive multinuclear osteoclast formation and (d) TRAP positive multinuclear osteoclast formation inhibition effect.

Figure 4, (a) TRAP activity inhibitory effect, (b) TRAP positive multinucleus of butanol fractions of Hwangnyeonhaedoktang (HRT) and Hwangnyeonhaedoktang fermented product (fHRT) under symbiotic culture conditions according to an embodiment of the present invention Results are shown for cell photos confirming the effect of inhibiting sexual osteoclast formation and (c) the inhibitory effect of TRAP positive multinucleated osteoclast formation.

Figure 5, (a) of the Hwangnyeon Haedoktang (HRT) and Hwangyeon Haedoktang fermentation product (fHRT), (b) of the Hwangyeon Haedoktang (HRT) and the Hwangyeon Haedangtang fermentation product (fHRT) according to an embodiment of the present invention It is a graph showing the effect of ethyl acetate fraction (EA) and (c) butanol fraction (BU) of Hwangnyeonhaedoktang (HRT) and Hwangnyeonhaedoktang fermentation (fHRT) on the cell growth.

Figure 6, (a) Hwangryunhaedeoktang butanol fraction (HRT-BU) and Hwangryanghaedeoktang fermented product 127 butanol fraction (fHRT-BU127) and (b) Hwangyeonhaengdeoktang butanol fraction (HRT) according to an embodiment of the present invention -BU) and Hwangnyeonhaedoktang fermentation 166 butanol fraction (fHRT-BU166) shows the effect on the MAPK and NF-kB signaling system.

Figure 7 is the Hwangnyeonhaedaktang butanol fraction (HRT-BU), the Hwangnyeonhaedoktang fermented product 127 butanol fraction (fHRT-BU127) and the Hwangnyeonhaedoktang fermented product 166 butanol fraction (fHRT-BU166) according to an embodiment of the present invention It shows the effect on the expression of NFATc1 and Atp6v0d2 protein.

FIG. 8 is a butanol fraction (HRT-BU), a 127 butanol fraction (fHRT-BU127) and a 166 butanol fraction of a fermentation product (fHRT-BU166) of Hwangnyeonhaedoktang according to an embodiment of the present invention. QPCR results are shown for mRNA expression of genes (A) ICAM-1, (B) NFkb2, (C) TNF-α and (D) I-kBα regulated by NF-kB.

Figure 9, Hwangnyeonhaedaktang butanol fraction (HRT-BU), Hwangnyeonhaedoktang fermented product 127 butanol fraction (fHRT-BU127) and Hwangnyeonhaedaktang fermented product 166 butanol fraction (fHRT-BU166) according to an embodiment of the present invention (A) TRAP activity inhibitory effect, (B) TRAP positive multinucleated osteoclast formation effect, and (C) Western blot results on BMM cells overexpressing NFATc1

FIG. 10 shows (a) bone mineral density (BMD), (b) bone volume / trabecular volume (BV / TV), and (c) bone surface / bone volume (BS) of an ovarian ablation rat according to an embodiment of the present invention. / BV), (d) Trabecular Thinckness (Tb.Th), (e) Trabecular separation (Tb.Sp) and (f) Trabecular number (Tb.N).

Figure 11 shows the femoral Micro-CT results of the ovarian ablation rat according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail by the following Preparation Examples and Examples. However, the following Preparation Examples and Examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example: Preparation of Lactobacillus Lactobacillus Ferment (fHRT)

1) Manufacture of HRT

250 g of yellow lotus, gold, yellow white, and gardenia were prepared, and a total of 1000 g was added to 10 l bottled water, and then deposited for 1 hour. Then, hot water was extracted for 180 minutes at 115 ° C. The extract was filtered using test sieve (106 ㎛) to obtain Hwangyeon Haedoktang.

2) Lactic Acid Bacteria Fermentation of Hwangnyeon Haedok-tang

Lactobacillus genus and Bifidobacterium genus were used from Korea Food Research Institute (KFRI) to prepare fHRT (Hwangnyunhaedoktang Lactic Acid Bacteria Fermentation Products) The fermented strains used to prepare are shown. After subcultured twice in MRS medium at 37 ° C. for 24 hours, the culture solution was injected again into the medium. In order to obtain a population of 1 × 10 ⁶ to 5 × 10 ⁶ CFU / ml, the cells were diluted appropriately and used as inoculum. For fermentation, 5 ml of HRT (Hwangryehaedangtang) prepared in Example 1) was incubated at 0.05 ml of the inoculum.

Table 1 KFRI No. Fermentation strain Fermented strain provider Fermented strain number and strain information 127 Lactobacillus casei KCTC 2180 128 Lactobacillus acidophilus KCTC 2182 129 Lactobacillus casei ATCC 393 144 Lactobacillus plantarum ATCC 8014 145 Lactobacillus fermentum ATCC 14931, Fermented beets 150 Lactobacillus acidophilus ATCC 4356IFO 13951, Human 161 Lactobacillus amylophilus NRRL B-4437 164 Lactobacillus fermentum NRRL B-4524 166 Lactobacillus curvatus NRRL B-4562 217 Lactobacillus acidophilus KCTC 3168, Human intestine 227 Lactobacillus confusus IH 14005 239 Lactobacillus brevis IH 14003 344 Lactobacillus bulgaricus - - 402 Lactobacillus plantarum ATCC 8014 442 Lactobacillus gelbruekiisubsp, lactis ATCC 7830IFO 3376 658 Lactobacillus gasseri NCFB 2233, DSM 20243, Human 692 Lactobacillus casei Yacurt - 693 Lactobacillus casei Yacurt - 744 Bifidobacterium breve ATCC 15700, Intestine of infant 748 Bifidobacterium thermophilum ATCC 25525, Swine feces 1092 Lactobacillus gasseri ATCC 33323 * NRRL: Norther Regional Research Laboratory, ARS Culture Collection, US Department of Agriculture, Peoria, III, USA * DSM: Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH, Braunschweig, Germany * NCFB: National collection of Food Bacteria, Reading United Kingdom * IFO: Insite for Fermentation, Osaka, Japan

Experimental Example 1: in-vitro analysis

1) TRAP Activity Assay

In order to confirm the efficacy of inhibiting the osteoclast differentiation marker TRAP enzyme activity, RAW264.7 cells differentiated into osteoclasts by RANKL were treated with HRT (Hwangryehaedoktang) and fHRT (Hanryanghaedoktang fermented product) and then TRAP. Activity was measured. Osteoclasts were obtained from bone marrow cells of mice. Bone marrow cells were incubated for 3 days in α-MEM consisting of 10% FBS containing M-CSF (60 ng / ml) to obtain bone marrow cell-derived macrophages (BMMs), which were used as osteoclast progenitor cells. For osteoclast production, BMMs were incubated with M-CSF (60 ng / ml) and RANKL (150 ng / ml) for 4 days. For osteoclast production from symbiotic cultures including osteoblasts and bone marrow cells, osteoblasts were prepared from the skull of newborn mice. Bone marrow cells (BMCs) and osteoblasts were co-cultured for 6 days in the presence of 1.25 (OH) ₂ D ₃ (1 × 10 ⁻⁸ M). Cultured cells were fixed and stained for measuring TRAP activity. TRAP activity was measured at 405 nm absorbance after treatment with the substrate (q-nitrophenyl phosphate, q-nitrophenyl phosphate).

① TRAP Enzyme Inhibitory Activity of HRT (Hwangryanghaedoktang) and fHRT (Hanryanghaedoktang Fermented Products)

100 μg / ml of Hwangyeon Haedaktang (HRT) and Hwangyeon Haedaktang Ferment (fHRT) prepared in Example were treated with RAW264.7 cells to analyze the effect of inhibiting TRAP activity, and the results are shown in FIG. 1. As shown in FIG. 1, it was confirmed that fHRT127 decreased TRAP activity by 13%, fHRT144 6%, and fHRT166 8% compared to HRT.

② TRAP enzyme inhibitory activity by concentrations of HRT (Hwangryanghaedoktang) and fHRT (Hanryanghaedoktang fermented product)

In order to determine the effect of the concentration, the treatment of fHRT127 and fHRT166 and HRT at a concentration of 12.5 to 200 ㎍ / ml to analyze the effect of TRAP activity inhibitory effect and TRAP positive multinucleated osteoclast formation, it is shown in FIG. As shown in FIG. 2, 50, 100 and 200 μg / ml HRT inhibited 19, 67 and 94% of TRAP enzyme activity. At 100 μg / ml, 11% of fHRT127 and 18% of fHRT166 were added as compared to HRT. TRAP activity inhibitory effect was shown. In addition, it was confirmed that HRT and two types of fHRTs at about 100 μg / ml showed about 90% of TRAP-positive multinucleated osteoclast formation.

③ Analysis of TRAP enzyme inhibitory activity and TRAP positive multinuclear osteoclast production of HRT (Hwangryeonhaedoktang) and fHRT (Hanryonhaedoktang fermented product) fractions

The HRT or fHRT prepared in the above Example was fractionated by using solvent extraction with ethyl acetate (EA) and n-butanol (BU), and each fraction was dried and stored in a -20 ° C dryer. TRAP enzyme inhibitory activity was analyzed after each fraction was treated with 0-30 ㎍ / ml, the results are shown in FIG.

As shown in FIG. 3, fHRT-EA127 (ethyl acetate fraction of Hwangryanghaedoktang fermented product 127) was 24% and fHRT-EA166 (10%) compared to HRT-EA (Hwangryanghaedoktang ethyl acetate fraction) at a concentration of 10 μg / ml. Ethyl acetate fraction of Hwangnyeonhaedoktang Ferment 166) showed a 9% TRAP activity inhibitory effect. In addition, HRT-BU (Hwangryanghaedoktang butanol fraction) showed TRAP inhibitory activity of 17%, 67% and 95%, respectively, at concentrations of 3, 10, and 30 μg / ml, compared to 0 μg / ml. Compared to the μg / ml HRT-BU, fHRT-BU127 (butanol fraction of Hwangryehaedoktang fermentation 127) was 23%, and fHRT-BU166 (butanol fraction of Hwangryehaedoktang fermentation 166) was 32% for additional TRAP inhibitory effect. Seemed. Compared with 10 μg / ml HRT-BU, 21% of fHRT-BU127 and 22% of fHRT-BU166 showed additional inhibitory effect, and 3% and fHRT- of fHRT-BU127 compared to 30 μg / ml HRT-BU. BU166 showed an additional 3% inhibitory effect on TRAP activity.

In addition, 3, 10, 30 μg / ml HRT-BU showed 55%, 97%, and 98% of TRAP-positive multinucleated osteoclast formation inhibitory effect compared to 0 μg / ml, and 3 μg / ml HRT- Compared with BU, fHRT-BU127 and fHRT-BU166 showed 34% additional TRAP positive multinuclear osteoclast formation inhibitory effect.

④ Inhibitory effect of HRT-BU and fHRT-BU on TRAP activity and TRAP-positive multinucleated osteoclast formation under symbiotic conditions

To determine the effects of HRT-BU and fHRT-BU on TRAP enzyme activity and TRAP-positive multinucleated osteoclast formation under conditions of bone marrow and pre-osteeoblast co-culture, the HRT-BU and fHRT-BU Was analyzed by 0 to 30 ㎍ / ml, the results are shown in FIG. As shown in FIG. 4, 10 and 30 μg / ml of HRT-BU compared to 0 μg / ml showed 53 and 95% of TRAP activity inhibitory efficacy, and fHRT- compared to 10 μg / ml HRT-BU. BU127 showed an additional inhibitory activity of 24% and fHRT-BU166 of 33%. In addition, fHRT-BU127 and fHRT-BU166 showed an additional 3% inhibitory effect on TRAP activity compared to 30 μg / ml HRT-BU.

In addition, 3, 10, 30 μg / ml HRT-BU showed 34%, 96% and 98% of TRAP-positive multinucleated osteoclast formation inhibition effect compared to 0 μg / ml, and 3 μg / ml HRT-BU. In comparison, fHRT-BU127 and fHRT-BU166 showed 34% additional TRAP positive multinucleated osteoclast formation inhibitory effect, respectively. Compared with 10 μg / ml HRT-BU, each showed 3% additional TRAP-positive multinucleated osteoclast formation inhibitory effect.

2) cell viability assay

BMM cells (1 × 10 ⁴ cell / well) were incubated with samples at various concentrations (12.5 to 200 μg / ml) for 3 days in 96-well plates in the presence of M-CSF (60 ng / ml). Cell counting Kit-8 (CCK-8) was used to measure cell viability, and the data were expressed as mean and standard error of three replicates. As shown in Figure 5, it was confirmed that the HRT (Hwangryanghaedoktang), fHRT (Hanryanghaedoktang fermented product) used in the experiment did not inhibit the growth. At 10 μg / ml, HRT-EA did not inhibit cell growth, but fHRT-EA127 (ethyl acetate fraction of Hwangyeonhadoktang fermented product 127) was 26%, fHRT-EA166 ( Ethyl acetate fraction of Hwangnyeonhaedoktang fermentation 166) inhibited further cell growth by 22%, compared with 30 ㎍ / ml HRT-EA, 35% for fHRT-EA127 and 36% for fHRT-EA166. Suppressed. On the other hand, fHRT-BU (butanol fraction of Hwangryonghaedaktang fermentation) did not show cell growth inhibitory activity.

Therefore, according to the results of 1) and 2) of Experimental Example 1, HRT-BU and fHRT-BU fractions having no cytotoxicity and additional TRAP activity inhibitory efficacy were used in other in-vitro experiments.

3) Western blot analysis

BMMs were washed with cold phosphate buffered saline (PBS) and washed with 50 mM tris-hydrochloric acid (pH 8.0), 5 mM EDTA, 150 mM sodium chloride, 1% NP-40, 0.1% SDS, 1 nM PMSF, lyase inhibitor tablets and It was dissolved in proteinn extraction buffer consisting of phosphatase inhibitor mixed tablets. Cell lysates were centrifuged at 10,000 × g for 15 minutes at 4 ° C. Protein concentration was measured using the BCA protein assay kit, and protein samples (20 μg) were sample buffer (100 nM tris-hydrochloric acid, 2% SDS, 1% 2-mecaptoethanol, 2% glycerol and 0.01% bromophenol Blue, pH 7.6), incubated at 95 ° C. for 5 minutes and dropwise onto a 12% polyacrylamine gel. Electrophoresis was performed using mini protean 3 cells (Bio-Rad, Hercules, CA, USA), and proteins separated on the gel were transferred to PVDF membranes. Membranes were incubated in blocking buffer (10 mM tris-hydrochloric acid (pH 7.5), 150 mM sodium chloride, 0.1% Tween 20 and 3% non-fat dry milk) and then at room temperature with primary antibody diluted 1: 1000. Incubated for 2 hours. After washing three times for 10 minutes each with wash buffer (10 mM tris-hydrochloric acid (pH 7.5), 150 mM sodium chloride and 0.1% Tween 20), the secondary antibody diluted 1: 2000 (Cell Signaling Technology Inc., Danvers) , MA, USA) and incubated for 1 hour. The membrane was then washed three times for 10 minutes each with wash buffer (10 nM tris-hydrochloric acid (pH 7.5), 150 mM sodium chloride and 0.1% Tween 20) before using SuperSignal West Femto Maximum Sensitivity Substrate. Chemiluminescent signals were detected by LAS-3000 Luminescent image analyzer.

(1) Effect of HRT-BU (Hwangryeon Haedaktang Butanol Fraction) and fHRT-BU (Hwangryeon Haedaktang Fermented Product Butanol Fraction) on MAPK and NF-kB Signaling System

The effects of the fractions of HRT (Hwangryanghaedeoktang) and fHRT (Hwangryonhaedeoktang fermentation) on the MAPK and NF-kB signaling system associated with osteoclast differentiation were confirmed, and the results are shown in FIG. 6. As shown in FIG. 6, fHRT-BU127 further inhibited the activity of p38, IKKα / β, and p65 increased by RANKL treatment compared to HRT-BU, and fHRT-BU166 further inhibited p-ERK and p-JNK. It was confirmed that.

(2) Effects of HRT-BU (butanol fraction of Hwangryunhaedeoktang) and fHRT-BU (butanol fraction of fermented product of Hwangryunhaedeoktang) on NFATc1 and Atp6v0d2 expression

The effect on the expression of Atp6v0d2 involved in osteoclast differentiation-related master transcription factors NFATc1 and multinuclear osteoclast formation was shown, and the results are shown in FIG. 7. As shown in FIG. 7, it was confirmed that protein expression of NFATc1 and Atp6v0d2 was increased at the 2-3 days of RANKL treatment, respectively, and HRT-BU (10 μg / ml) inhibited the increase in expression of both proteins and HRT-BU. It was confirmed that fHRT-BU127 (10 μg / ml) and fHRT-BU166 (10 μg / ml) additionally inhibited the expression of two proteins.

4) real-time quantitative PCR (QPCR) analysis

To assess the efficacy of mRNA expression of genes regulated by NF-kB, cells (3 × 10 ⁵ cell / well in a 6 well plate) were sampled for 2 hours in the presence of M-CSF (60 ng / ml). Incubated with. Total RNA was isolated by RNA easy mini kit after 3, 6, 12 hours of RANKL (150 ng / ml). First-strand cDNA was synthesized with 1 μg total RNA, 1 μM oligo-dT ₁₈ primer, RNase 10 units and Omniscript Reverse Transcriptase. Subsequently, SYBR green-based QPCR amplification was performed using SYBR Green PCR Master Mix and Applied Biosystems 7500 Real-time PCR system using first-strand cDNA and 10 pmol primer diluted 1: 3. The PCR reaction consists of three stages, the first being polymerase activation for 10 minutes at 95 ℃. The second step carries out a three step cycle for 30 seconds at 94 ° C, 40 seconds at 60 ° C and 1 minute at 72 ° C. The third step for generating the PCR product Temperature melting curve is performed for 1 minute at 95 ℃, 30 seconds at 60 ℃ and 30 seconds at 95 ℃. All reactions were repeated three times, and data were analyzed by the 2 ^-ΔΔCT method. β-actin was used as an internal standard. The analysis results are shown in FIG. 8.

As shown in FIG. 8, it was confirmed that HRT-BU127 further inhibits gene mRNA expression of ICAM, TNF-a and Nfkb2 as compared to Vehicle.

5) Effect of Fractions of HRT (Hwangnyeon Hae-Dok-tang) and fHRT (Hwang-Ryeon Hae-Dok-tang Fermentation) on NFATc1 Overexpression

Ectopic expression of transcription factor NFATc1 in osteoclasts was investigated, and it was investigated whether it could overcome the differentiation inhibitory effect of HRT-BU (butanol fraction of Hwangryehaedoktang) and fHRT-BU (butanol fraction of fermented Hwanghaehaedoktang). DNA fragments from MSCV-CaNFATc1 encoding HA-tagged Ca-NFATc1 (the Xhol / Klenow-EcoRI fragment) were subcloned with pMX-puronow-EcoRI together with the puromycin selection factor (pMX-Ca-NFATc1). To prepare retroviral stocks, pMX vectors (control) and pMX-Ca-NFATc1 retroviral vectors were injected into PLAT-E cells using LiPofectamine 2000 (Invitrogen). The medium was obtained after 2 days, and further incubated with M-CSF (120 ng / ml) and polybrene (5 μg / ml) in BMMs (1 × 10 ⁷ cell / 100 mm dish) for 2 days. . Puromycin-resistant BMMs differentiated into osteoclasts for 4-5 days in the presence of 60 ng / ml M-CSF and 150 ng / ml RANKL. The investigation result is shown in FIG. As shown in FIG. 9, HRT-BU showed 78%, fHRT-BU127 88% and fHRT-BU166 90% TRAP inhibitory activity in the BMM transfected with pMX vector, HRT-BU showed 93%, fHRT-BU127 showed 98% and fHRT-BU166 showed 95% TRAP-positive multinucleated osteoclast formation. In addition, compared to the pMX vector, BMM trasfection with Ca-NFATc1 showed about two-fold increase in TRAP activity, compared with BMM trasnfection with Ca-NFATc1, HRT-BU was 85%, fHRT-BU127 was 90% and fHRT-BU166 showed 95% of the inhibitory effect on TRAP activity. In addition, compared with the pMX vector, BMM transfected with Ca-NFATc1 increased the formation of multinucleated osteoclasts by about three times.HRT-BU was 92% and fHRT-BU127 was 88 compared to BMM transfected with Ca-NFATc1. % And fHRT-BU166 showed 98% of TRAP-positive multinucleated osteoclast formation inhibition effect.

Experimental Example 2: in-vivo  analysis

1) Experimental Animal

190-210 g of 8 week old female Sprague-Dawley rats (Orient Bio Inc., Seoul, Korea) were used for the experiment. Before the experiment, rats were used for the experiment after 7 days of acclimation. During the acclimation process, rats were reared freely in the control room with 22 ± 1 ℃ temperature and 55 ± 10% humidity. Thereafter, sham-operate (Sham, n = 8) or ovarian resection (OVX, n = 56) was performed. Animal experiments were handled in accordance with the Korean Experimental Research Regulations.

2) Analyze bone loss effect according to sample administration and sample

The ovarian ablation (OVX) rats were divided into 7 groups of 8 rats one week after surgery, and two concentrations (0.3 g / kg and 1.0) of Hwangnyeonhaedoktang (HRT) and Hwangnyeonhaedoktang fermented products (fHRT127 and fHRT166) for 3 months. g / kg) was orally administered, and the detailed experimental group is shown in Table 2 below.

TABLE 2 group Sample dose OVX Bidirectional OVX HRT-0.3 0.3 g / kg HRT injection after bidirectional OVX HRT-1.0 1.0 g / kg HRT injection after bidirectional OVX fHRT127-0.3 0.3 g / kg fHRT127 injection after bidirectional OVX fHRT127-1.0 1.0 g / kg fHRT127 injection after bidirectional OVX fHRT166-0.3 0.3 g / kg fHRT166 injection after bidirectional OVX fHRT166-1.0 1.0 g / kg fHRT166 injection after bidirectional OVX

The effect on bone loss in the femur of ovarian ablation (OVX) rats according to the sample administration is shown in FIG. 10. As shown in Figure 10, compared with the Sham group, OVX group has a 70% reduction in BMD, bone volume / trabecular volume (BV / TV) 65% reduction, bone surface / bone volume raito (BS / BV) 29% increase, 14% decrease in Trabcular Thinckness (Tb.Th), 169% increase in Trabecular separation (Tb.Sp), and 60% decrease in Trabecular number (Tb.N). The results are shown in more detail through 10A to 10F.

 As shown in FIG. 10A, the Sham group was 100%, and as a result, fHRT127-1.0 was 55% and fHRT166-0.3 was 52%, and BMD was significantly increased as compared to the OVX group. HRT-0.3 In comparison, fHRT166-0.3 was 43%, and fHRT127-1.0 was 54% compared to HRT-1.0.

As shown in FIG. 10B, the BV / TV was significantly increased by 35% for fHRT127-1.0 and 31% for fHRT166-0.3 compared to OVX group, and 27% of fHRT166-0.3 compared to HRT-0.3. BV / TV was increased but not significant. In addition, fHRT127-1.0 significantly increased BV / TV by 41% compared to HRT-1.0.

As shown in FIG. 10C, BS / BV significantly decreased to 34% for fHRT127-1.0, 34% for fHRT166-0.3 and 20% for fHRT166-1.0 compared to the OVX group. In addition, compared to HRT-0.3 and HRT-1.0, fHRT166-0.3 and fHRT127-1.0 were significant by 23%, respectively, and further reduced BS / BV.

As shown in FIG. 10D, compared to the OVX group, fHRT127-1.0 increased 21%, fHRT166-0.3 increased 19%, and fHRT166-1.0 increased 11%, and Hb-0.3 and HRT- significantly increased. Compared with 1.0, fHRT166-0.3 and fHRT127-1.0 were 15% significant and additionally increased Tb.Th.

As shown in FIG. 10E and FIG. 10F, no significant changes in Tb.Sp and Tb.N were found compared to the OVX group.

2) Mitro-computed tomography (Micro-CT) analysis

For 3D skeletal structure analysis, histomorphologic analysis was performed with an 8 μm resolution eXplore Locus scanner (GEHealthcare UK Ltd., Buckinghamshire, UK). All morphometric parameters were calculated using eXplore Micro-View (wersion 2.2, GEHealthcare). In the femur, the scanner area was confined to a peripheral interosseous tip about 1.7 mm extended from the initial cavernous proximal tip, and the results are shown in FIG. 11.

Statistical analysis of all the above experiments was performed using SPSS software. TRAP activity of osteoclasts and significant mRNA expression level of the relevant gene is β- ektin (actin) -normalized 2 ^- was calculated as studen's t-test with ^{△△ CT} value, for statistical analysis of the animal experiments, parametric of the dispersion One-way analysis was used for all other group tests. The Duncan multiple comparison test was used to identify significant differences between the mean values and p values lower than 0.05 were considered to be meaningful.

Claims (12)

Pharmaceutical composition for the prevention or treatment of bone diseases, including the lactic acid bacteria fermentation product of Hwangnyeonhaedoktang as an active ingredient. The pharmaceutical composition according to claim 1, wherein the Hwangnyeon Haedoktang is a hot water extract of Hwangyeon, Golden, Hwangbaek and Gardenia. The pharmaceutical composition according to claim 1, wherein the lactic acid bacteria fermented product is inoculated with lactic acid bacteria to Hwangnyeonhaedoktang and fermented under an aerobic atmosphere at a temperature of 35 ° C to 45 ° C for a time of 1 day to 3 days. . According to claim 3, wherein the lactic acid bacteria Lactobacillus (Lactobacillus) or Bifidobacterium (Bifidobacterium) genus pharmaceutical composition, characterized in that the genus. According to claim 4, The Lactobacillus (Lactobacillus) genus, Lactobacillus casei (Lactobacillus casei), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus fermentum (Lactobacillus) fermentum), Lactobacillus curvatus, Lactobacillus confusus and Lactobacillus gasseri (Lactobacillus gasseri) pharmaceutical composition, characterized in that at least one member selected from the group consisting of. The pharmaceutical composition according to claim 4, wherein the Bifidobacterium genus is a Bifidobacterium breve or Bifidobacterium thermophilum strain. Health functional food composition for the prevention or improvement of bone disease containing lactic acid bacteria fermentation product of Hwangnyeonhaedoktang as an active ingredient. According to claim 7, wherein the Hwangnyeonhaedoktang health functional food composition, characterized in that the hot water extracts of yellow, golden, yellow and yellow. The health functional food according to claim 7, wherein the lactic acid bacteria fermented product is inoculated with lactic acid bacteria to the Hwangnyeonhaedoktang and fermented under an aerobic atmosphere at a temperature of 35 ° C to 45 ° C for a period of 1 day to 3 days. Composition. The health functional food composition according to claim 9, wherein the lactic acid bacteria is Lactobacillus or Bifidobacterium. The method of claim 10, wherein the genus Lactobacillus, Lactobacillus casi (Lactobacillus casei), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus permentum (Lactobacillus) fermentum), Lactobacillus curvatus (Lactobacillus curvatus), Lactobacillus confusus (Lactobacillus confusus) and Lactobacillus gasseri (Lactobacillus gasseri) health functional food composition, characterized in that at least one member selected from the group consisting of. The Bifidobacterium genus is a Bifidobacterium breve or Bifidobacterium thermophilum strain, characterized in that the health functional food composition.

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