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WO2013176327A1 - Composition pharmaceutique et aliment fonctionnel de santé pour la prévention ou le traitement de maladies osseuses, contenant un produit de fermentation de bactéries lactiques de soupe détox de coptis chinensis en tant que principe actif - Google Patents

Composition pharmaceutique et aliment fonctionnel de santé pour la prévention ou le traitement de maladies osseuses, contenant un produit de fermentation de bactéries lactiques de soupe détox de coptis chinensis en tant que principe actif Download PDF

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Publication number
WO2013176327A1
WO2013176327A1 PCT/KR2012/004290 KR2012004290W WO2013176327A1 WO 2013176327 A1 WO2013176327 A1 WO 2013176327A1 KR 2012004290 W KR2012004290 W KR 2012004290W WO 2013176327 A1 WO2013176327 A1 WO 2013176327A1
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Prior art keywords
lactobacillus
lactic acid
acid bacteria
hrt
hwangnyeonhaedoktang
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Korean (ko)
Inventor
마진열
조원경
송광훈
김태수
심기석
하현일
정다영
송석영
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Korea Institute of Oriental Medicine KIOM
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Korea Institute of Oriental Medicine KIOM
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • A61K36/718—Coptis (goldthread)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/539—Scutellaria (skullcap)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs

Definitions

  • 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.
  • Bone calcification does not occur when RUNX2, which is an important transcription factor in osteoblasts, decreases or the expression level decreases.
  • RUNX2 which is an important transcription factor in osteoblasts, decreases or the expression level decreases.
  • RANKL receptor activator of NF-kB ligand
  • OPG Ostoprotegrin
  • 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.
  • Osteoblasts that produce bone
  • Osteoblasts produce RANKL and its inducing receptor, OPG.
  • OPG osteoclast progenitor receptor
  • RANKL a receptor on the surface of osteoclast progenitor cells
  • osteoclast progenitor cells mature into osteoclasts, resulting in bone resorption.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • a pharmaceutical composition for the prevention or treatment of bone diseases comprising the lactic acid bacteria fermentation product of Hwangnyeonhaedoktang as an active ingredient.
  • 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.
  • 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 °C to 40 °C and 1 to 3 days.
  • the lactic acid bacteria is preferably Lactobacillus (Lactobacillus) or Bifidobacterium (Bifidobacterium) genus.
  • Lactobacillus 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.
  • '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.
  • the composition containing the lactic acid bacteria fermentation product of Hwangnyeonhaedoktang is effective in the prevention or treatment of bone diseases.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • oral preparations suppositories, or sterile injectable solutions, respectively, according to conventional methods.
  • 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.
  • excipients for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like in the lactic acid bacterium fermentation product of Hwangnyeonhaedoktang.
  • 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.
  • 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.
  • base of the suppository utopsol, macrosol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • 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.
  • 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.
  • 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.
  • HRT Hwangnyeonhaedoktang
  • fHRT Hwangnyeonhaedoktang fermented product
  • FIG. 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.
  • HRT Hwangnyeonhaedoktang
  • fHRT Hwangnyeonhaedoktang fermented product
  • FIG. 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.
  • EA ethyl acetate
  • HRT Hwangnyeonhaedaktang
  • fHRT Hwangnyeonhaedoktang fermented product
  • FIG. 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.
  • HRT Hwangnyeonhaedoktang
  • fHRT Hwangnyeonhaedoktang fermented product
  • FIG. 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.
  • EA ethyl acetate fraction
  • BU butanol fraction
  • FIG. 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- ⁇ and (D) I-kB ⁇ regulated by NF-kB.
  • Hwangnyeonhaedaktang butanol fraction HRT-BU
  • Hwangnyeonhaedoktang fermented product 127 butanol fraction fHRT-BU127
  • Hwangnyeonhaedaktang fermented product 166 butanol fraction fHRT-BU166
  • A TRAP activity inhibitory effect
  • B TRAP positive multinucleated osteoclast formation effect
  • 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.
  • BMD bone mineral density
  • BV bone volume / trabecular volume
  • BS bone surface / bone volume
  • 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 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 shows (a) bone mineral density (BMD), (b) bone volume / trabecular volume (
  • Figure 11 shows the femoral Micro-CT results of the ovarian ablation rat according to an embodiment of the present invention.
  • Lactobacillus genus and Bifidobacterium genus were used from Korea Food Research Institute (KFRI) to prepare fHRT (Hwangnyunhaedoktang Lactic Acid Bacteria Fermentation Products)
  • KFRI Korea Food Research Institute
  • the culture solution was injected again into the medium.
  • the cells were diluted appropriately and used as inoculum.
  • 5 ml of HRT (Hwangryehaedangtang) prepared in Example 1) was incubated at 0.05 ml of the inoculum.
  • 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 ⁇ -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.
  • BMMs bone marrow cell-derived macrophages
  • BMMs were incubated with M-CSF (60 ng / ml) and RANKL (150 ng / ml) for 4 days.
  • M-CSF 60 ng / ml
  • RANKL 150 ng / ml
  • 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).
  • HRT Hwangyeon Haedaktang
  • fHRT Hwangyeon Haedaktang Ferment
  • HRT Hewangryanghaedoktang
  • fHRT Heanryanghaedoktang fermented product
  • the treatment of fHRT127 and fHRT166 and HRT at a concentration of 12.5 to 200 ⁇ g / ml to analyze the effect of TRAP activity inhibitory effect and TRAP positive multinucleated osteoclast formation it is shown in FIG.
  • FIG. 2 50, 100 and 200 ⁇ g / ml HRT inhibited 19, 67 and 94% of TRAP enzyme activity.
  • 100 ⁇ g / ml 11% of fHRT127 and 18% of fHRT166 were added as compared to HRT.
  • TRAP activity inhibitory effect was shown.
  • HRT and two types of fHRTs at about 100 ⁇ g / ml showed about 90% of TRAP-positive multinucleated osteoclast formation.
  • 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 ⁇ g / ml, the results are shown in FIG.
  • 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 ⁇ g / ml.
  • Ethyl acetate fraction of Hwangnyeonhaedoktang Ferment 166) showed a 9% TRAP activity inhibitory effect.
  • HRT-BU Hewangryanghaedoktang butanol fraction
  • TRAP inhibitory activity 17%, 67% and 95%, respectively, at concentrations of 3, 10, and 30 ⁇ g / ml, compared to 0 ⁇ g / ml.
  • fHRT-BU127 butanol fraction of Hwangryehaedoktang fermentation 127)
  • fHRT-BU166 butanol fraction of Hwangryehaedoktang fermentation 166) was 32% for additional TRAP inhibitory effect. Seemed.
  • fHRT-BU127 Compared with 10 ⁇ g / 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 ⁇ g / ml HRT-BU. BU166 showed an additional 3% inhibitory effect on TRAP activity.
  • HRT-BU and fHRT-BU were analyzed by 0 to 30 ⁇ g / ml, the results are shown in FIG.
  • 10 and 30 ⁇ g / ml of HRT-BU compared to 0 ⁇ g / ml showed 53 and 95% of TRAP activity inhibitory efficacy
  • fHRT- compared to 10 ⁇ g / ml HRT-BU BU127 showed an additional inhibitory activity of 24% and fHRT-BU166 of 33%.
  • fHRT-BU127 and fHRT-BU166 showed an additional 3% inhibitory effect on TRAP activity compared to 30 ⁇ g / ml HRT-BU.
  • BMM cells (1 ⁇ 10 4 cell / well) were incubated with samples at various concentrations (12.5 to 200 ⁇ g / 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.
  • 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 ⁇ g / 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.
  • fHRT-BU butanol fraction of Hwangryonghaedaktang fermentation
  • HRT-BU and fHRT-BU fractions having no cytotoxicity and additional TRAP activity inhibitory efficacy were used in other in-vitro experiments.
  • 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 ⁇ g) 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.
  • blocking buffer 10 mM tris-hydrochloric acid (pH 7.5), 150 mM sodium chloride, 0.1% Tween 20 and 3% non-fat dry milk
  • 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 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.
  • HRT Hewangryanghaedeoktang
  • fHRT Hewangryonhaedeoktang fermentation
  • HRT-BU butanol fraction of Hwangryunhaedeoktang
  • fHRT-BU butanol fraction of fermented product of Hwangryunhaedeoktang
  • FIG. 7 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 ⁇ g / ml) inhibited the increase in expression of both proteins and HRT-BU. It was confirmed that fHRT-BU127 (10 ⁇ g / ml) and fHRT-BU166 (10 ⁇ g / ml) additionally inhibited the expression of two proteins.
  • 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 °C.
  • 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 °C, 30 seconds at 60 °C and 30 seconds at 95 °C. All reactions were repeated three times, and data were analyzed by the 2 - ⁇ CT method. ⁇ -actin was used as an internal standard. The analysis results are shown in FIG. 8.
  • HRT-BU127 further inhibits gene mRNA expression of ICAM, TNF-a and Nfkb2 as compared to Vehicle.
  • 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 ⁇ g / 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.
  • 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%
  • fHRT-BU166 showed 95% TRAP-positive multinucleated osteoclast formation.
  • BMM trasfection with Ca-NFATc1 showed about two-fold increase in TRAP activity
  • HRT-BU was 85%
  • fHRT-BU127 was 90% and fHRT-BU166 showed 95% of the inhibitory effect on TRAP activity.
  • 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.
  • 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).
  • BMD bone volume / trabecular volume
  • BS / BV bone surface / bone volume raito
  • Tb.Th Trabcular Thinckness
  • Tb.Sp 169% increase in Trabecular separation
  • Tb.N 60% decrease in Trabecular number
  • 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.
  • 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.
  • fHRT127-1.0 significantly increased BV / TV by 41% compared to HRT-1.0.
  • 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.
  • fHRT166-0.3 and fHRT127-1.0 were significant by 23%, respectively, and further reduced BS / BV.
  • fHRT127-1.0 increased 21%
  • fHRT166-0.3 increased 19%
  • fHRT166-1.0 increased 11%
  • fHRT166-0.3 and fHRT127-1.0 were 15% significant and additionally increased Tb.Th.

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103735719A (zh) * 2014-01-12 2014-04-23 万世凤 一种复方黄连胶囊制作方法
CN117279521A (zh) * 2021-05-17 2023-12-22 好侍健康食品株式会社 用于抑制破骨前体细胞分化为破骨细胞的组合物及用于改善骨代谢的组合物

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019050063A1 (fr) * 2017-09-07 2019-03-14 태이생명과학 주식회사 Composition pour prévenir ou traiter des maladies parodontales comprenant, en tant que principe actif, un extrait de scutellaria baicalensis traité avec une enzyme isolée à partir d'aspergillus niger
KR102144838B1 (ko) * 2019-04-18 2020-08-14 전남대학교산학협력단 락토바실러스 쿠르바투스 WiKim38 균주 또는 이의 배양액을 포함하는 골 질환 개선, 예방 또는 치료용 조성물
KR102199934B1 (ko) * 2019-06-26 2021-01-08 한국식품연구원 황련해독탕의 김치유산균 발효물을 함유하는 여드름 개선용 화장료 조성물
KR102350437B1 (ko) * 2019-06-26 2022-01-14 한국식품연구원 황련해독탕의 김치유산균 발효물을 함유하는 여드름 개선용 화장료 조성물
KR102850602B1 (ko) * 2023-01-19 2025-08-26 재단법인 대구경북첨단의료산업진흥재단 골 질환 예방 또는 치료를 위한 신규 유산균 및 이의 배양액

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100700480B1 (ko) * 2006-02-24 2007-03-28 경희대학교 산학협력단 황백 추출물을 포함하는 골절의 예방 및 치료용 조성물
KR100740565B1 (ko) * 2006-01-25 2007-07-18 계명대학교 산학협력단 파골세포 분화억제 효과를 가지는 황금 추출물 및 이를 포함하는 건강기능식품 조성물
KR20110041677A (ko) * 2009-10-16 2011-04-22 안국약품 주식회사 치주질환 예방 또는 치료용 생약 조성물
KR20110139472A (ko) * 2010-06-23 2011-12-29 한국 한의학 연구원 방풍통성산 또는 이의 유산균 발효물을 포함하는 골다공증 예방 또는 치료용 조성물

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100740565B1 (ko) * 2006-01-25 2007-07-18 계명대학교 산학협력단 파골세포 분화억제 효과를 가지는 황금 추출물 및 이를 포함하는 건강기능식품 조성물
KR100700480B1 (ko) * 2006-02-24 2007-03-28 경희대학교 산학협력단 황백 추출물을 포함하는 골절의 예방 및 치료용 조성물
KR20110041677A (ko) * 2009-10-16 2011-04-22 안국약품 주식회사 치주질환 예방 또는 치료용 생약 조성물
KR20110139472A (ko) * 2010-06-23 2011-12-29 한국 한의학 연구원 방풍통성산 또는 이의 유산균 발효물을 포함하는 골다공증 예방 또는 치료용 조성물

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YANG, HYUN JU ET AL.: "Anti-inflammatory effects of Hwangnyeonhaedok-tang and Fermented Hwangnyeonhaedok-tang", JOURNAL OF KOREAN ORIENTAL MEDICAL OPHTHALMOLOGY & OTOLARYNGOLOGY & DERMATOLOGY, vol. 24, no. 2, August 2011 (2011-08-01), pages 1 - 15, XP053019200 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103735719A (zh) * 2014-01-12 2014-04-23 万世凤 一种复方黄连胶囊制作方法
CN117279521A (zh) * 2021-05-17 2023-12-22 好侍健康食品株式会社 用于抑制破骨前体细胞分化为破骨细胞的组合物及用于改善骨代谢的组合物
EP4342535A4 (fr) * 2021-05-17 2025-04-23 House Wellness Foods Corporation Composition pour l'inhibition de la différenciation de cellules précurseurs d'ostéoclastes en ostéoclastes, et composition pour améliorer le métabolisme osseux

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