KR101402926B1 - Pharmaceutical composition and functional food for prevention or treatment of bone disease comprising the lactic acid bacteria ferment of hwangryunhaedoktang - Google Patents
Pharmaceutical composition and functional food for prevention or treatment of bone disease comprising the lactic acid bacteria ferment of hwangryunhaedoktang Download PDFInfo
- Publication number
- KR101402926B1 KR101402926B1 KR1020120056355A KR20120056355A KR101402926B1 KR 101402926 B1 KR101402926 B1 KR 101402926B1 KR 1020120056355 A KR1020120056355 A KR 1020120056355A KR 20120056355 A KR20120056355 A KR 20120056355A KR 101402926 B1 KR101402926 B1 KR 101402926B1
- Authority
- KR
- South Korea
- Prior art keywords
- fermented
- lactobacillus
- bone
- fermented product
- hrt
- Prior art date
Links
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 241000894006 Bacteria Species 0.000 title claims abstract description 20
- 239000004310 lactic acid Substances 0.000 title claims abstract description 19
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 19
- 208000020084 Bone disease Diseases 0.000 title claims abstract description 17
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 10
- 235000013376 functional food Nutrition 0.000 title claims description 14
- 230000002265 prevention Effects 0.000 title description 2
- 239000009837 hwangryunhaedok-tang Substances 0.000 title 1
- 239000004480 active ingredient Substances 0.000 claims abstract description 7
- 239000000284 extract Substances 0.000 claims abstract description 6
- 210000000988 bone and bone Anatomy 0.000 claims description 29
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 22
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 21
- 208000001132 Osteoporosis Diseases 0.000 claims description 18
- 241000186660 Lactobacillus Species 0.000 claims description 14
- 229940039696 lactobacillus Drugs 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 14
- 238000000855 fermentation Methods 0.000 claims description 12
- 230000004151 fermentation Effects 0.000 claims description 12
- 230000036541 health Effects 0.000 claims description 12
- 244000199866 Lactobacillus casei Species 0.000 claims description 9
- 235000013958 Lactobacillus casei Nutrition 0.000 claims description 9
- 229940017800 lactobacillus casei Drugs 0.000 claims description 9
- 241001474374 Blennius Species 0.000 claims description 8
- 240000006024 Lactobacillus plantarum Species 0.000 claims description 6
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims description 6
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 6
- 229940072205 lactobacillus plantarum Drugs 0.000 claims description 6
- 239000011707 mineral Substances 0.000 claims description 6
- 235000010755 mineral Nutrition 0.000 claims description 6
- 208000010392 Bone Fractures Diseases 0.000 claims description 5
- 206010017076 Fracture Diseases 0.000 claims description 5
- 244000068988 Glycine max Species 0.000 claims description 5
- 235000010469 Glycine max Nutrition 0.000 claims description 5
- 241001134659 Lactobacillus curvatus Species 0.000 claims description 4
- 230000037182 bone density Effects 0.000 claims description 4
- 229940093499 ethyl acetate Drugs 0.000 claims description 4
- 235000019439 ethyl acetate Nutrition 0.000 claims description 4
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 3
- 239000012298 atmosphere Substances 0.000 claims description 3
- 235000013312 flour Nutrition 0.000 claims description 3
- 208000028169 periodontal disease Diseases 0.000 claims description 3
- 229940072033 potash Drugs 0.000 claims description 3
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 3
- 235000015320 potassium carbonate Nutrition 0.000 claims description 3
- 241000233614 Phytophthora Species 0.000 claims description 2
- 244000273928 Zingiber officinale Species 0.000 claims description 2
- 235000006886 Zingiber officinale Nutrition 0.000 claims description 2
- 235000008397 ginger Nutrition 0.000 claims description 2
- 239000012046 mixed solvent Substances 0.000 claims 2
- 241001105098 Angelica keiskei Species 0.000 claims 1
- 206010031243 Osteogenesis imperfecta Diseases 0.000 claims 1
- 229930014626 natural product Natural products 0.000 abstract description 2
- 208000035896 Twin-reversed arterial perfusion sequence Diseases 0.000 description 48
- 230000000694 effects Effects 0.000 description 44
- 210000002997 osteoclast Anatomy 0.000 description 41
- 230000002401 inhibitory effect Effects 0.000 description 35
- 230000015572 biosynthetic process Effects 0.000 description 24
- 210000004027 cell Anatomy 0.000 description 21
- 239000002034 butanolic fraction Substances 0.000 description 15
- 230000001965 increasing effect Effects 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 11
- 230000005764 inhibitory process Effects 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 11
- 210000000963 osteoblast Anatomy 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 210000004979 bone marrow derived macrophage Anatomy 0.000 description 8
- 230000003247 decreasing effect Effects 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 240000001046 Lactobacillus acidophilus Species 0.000 description 7
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 description 7
- 102100034404 Nuclear factor of activated T-cells, cytoplasmic 1 Human genes 0.000 description 7
- 101710151542 Nuclear factor of activated T-cells, cytoplasmic 1 Proteins 0.000 description 7
- 230000004069 differentiation Effects 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 7
- 229940039695 lactobacillus acidophilus Drugs 0.000 description 7
- 241000700159 Rattus Species 0.000 description 6
- 230000002457 bidirectional effect Effects 0.000 description 6
- 210000002798 bone marrow cell Anatomy 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 5
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 239000002038 ethyl acetate fraction Substances 0.000 description 5
- 239000000546 pharmaceutical excipient Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 101150027145 ATP6V0D2 gene Proteins 0.000 description 4
- 108010057466 NF-kappa B Proteins 0.000 description 4
- 102000003945 NF-kappa B Human genes 0.000 description 4
- 241000233622 Phytophthora infestans Species 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 241000186000 Bifidobacterium Species 0.000 description 3
- 208000006386 Bone Resorption Diseases 0.000 description 3
- 206010065687 Bone loss Diseases 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- 241000186840 Lactobacillus fermentum Species 0.000 description 3
- 108091054455 MAP kinase family Proteins 0.000 description 3
- 102000043136 MAP kinase family Human genes 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 206010037660 Pyrexia Diseases 0.000 description 3
- 244000299461 Theobroma cacao Species 0.000 description 3
- 108091023040 Transcription factor Proteins 0.000 description 3
- 102000040945 Transcription factor Human genes 0.000 description 3
- 230000024279 bone resorption Effects 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 229940011871 estrogen Drugs 0.000 description 3
- 239000000262 estrogen Substances 0.000 description 3
- 239000000796 flavoring agent Substances 0.000 description 3
- 235000013373 food additive Nutrition 0.000 description 3
- 239000002778 food additive Substances 0.000 description 3
- 229940012969 lactobacillus fermentum Drugs 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000011164 ossification Effects 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 230000011664 signaling Effects 0.000 description 3
- 210000000689 upper leg Anatomy 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 241001468229 Bifidobacterium thermophilum Species 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 208000032843 Hemorrhage Diseases 0.000 description 2
- 241000186606 Lactobacillus gasseri Species 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 241001165494 Rhodiola Species 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 241000186675 Weissella confusa Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 239000003125 aqueous solvent Substances 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 238000003570 cell viability assay Methods 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 235000010980 cellulose Nutrition 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 235000019219 chocolate Nutrition 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 235000015203 fruit juice Nutrition 0.000 description 2
- 230000000855 fungicidal effect Effects 0.000 description 2
- 239000000417 fungicide Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 241000411851 herbal medicine Species 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000010603 microCT Methods 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- -1 olive oil Chemical compound 0.000 description 2
- 210000004663 osteoprogenitor cell Anatomy 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 239000000454 talc Substances 0.000 description 2
- 229910052623 talc Inorganic materials 0.000 description 2
- 235000012222 talc Nutrition 0.000 description 2
- 239000002562 thickening agent Substances 0.000 description 2
- 210000001835 viscera Anatomy 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000516950 Atheroides Species 0.000 description 1
- 206010003645 Atopy Diseases 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 235000016068 Berberis vulgaris Nutrition 0.000 description 1
- 241000335053 Beta vulgaris Species 0.000 description 1
- 241000186012 Bifidobacterium breve Species 0.000 description 1
- 241000741973 Bifidobacterium breve DSM 20213 = JCM 1192 Species 0.000 description 1
- 229940122361 Bisphosphonate Drugs 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102000055006 Calcitonin Human genes 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 240000008574 Capsicum frutescens Species 0.000 description 1
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 description 1
- 102000015775 Core Binding Factor Alpha 1 Subunit Human genes 0.000 description 1
- 108010024682 Core Binding Factor Alpha 1 Subunit Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 208000000616 Hemoptysis Diseases 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102000015271 Intercellular Adhesion Molecule-1 Human genes 0.000 description 1
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 description 1
- SFBODOKJTYAUCM-UHFFFAOYSA-N Ipriflavone Chemical compound C=1C(OC(C)C)=CC=C(C2=O)C=1OC=C2C1=CC=CC=C1 SFBODOKJTYAUCM-UHFFFAOYSA-N 0.000 description 1
- 241000186714 Lactobacillus amylophilus Species 0.000 description 1
- 240000001929 Lactobacillus brevis Species 0.000 description 1
- 235000013957 Lactobacillus brevis Nutrition 0.000 description 1
- 244000199885 Lactobacillus bulgaricus Species 0.000 description 1
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 description 1
- 241001070017 Lactobacillus fermentum ATCC 14931 Species 0.000 description 1
- 241001662087 Lactobacillus gasseri ATCC 33323 = JCM 1131 Species 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010027304 Menopausal symptoms Diseases 0.000 description 1
- 206010027452 Metastases to bone Diseases 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 240000004371 Panax ginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 229920002230 Pectic acid Polymers 0.000 description 1
- 241000364057 Peoria Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 238000010818 SYBR green PCR Master Mix Methods 0.000 description 1
- 241000269821 Scombridae Species 0.000 description 1
- 206010039793 Seborrhoeic dermatitis Diseases 0.000 description 1
- 102000003800 Selectins Human genes 0.000 description 1
- 108090000184 Selectins Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 235000005764 Theobroma cacao ssp. cacao Nutrition 0.000 description 1
- 235000005767 Theobroma cacao ssp. sphaerocarpum Nutrition 0.000 description 1
- 102400000336 Thyrotropin-releasing hormone Human genes 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 102100028787 Tumor necrosis factor receptor superfamily member 11A Human genes 0.000 description 1
- 101710178436 Tumor necrosis factor receptor superfamily member 11A Proteins 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000001548 androgenic effect Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000000767 anti-ulcer Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 150000004663 bisphosphonates Chemical class 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000018678 bone mineralization Effects 0.000 description 1
- 230000010478 bone regeneration Effects 0.000 description 1
- 230000010072 bone remodeling Effects 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 235000001046 cacaotero Nutrition 0.000 description 1
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 0.000 description 1
- 239000001390 capsicum minimum Substances 0.000 description 1
- 235000014171 carbonated beverage Nutrition 0.000 description 1
- 210000003321 cartilage cell Anatomy 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 235000013985 cinnamic acid Nutrition 0.000 description 1
- 229930016911 cinnamic acid Natural products 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000002591 computed tomography Methods 0.000 description 1
- 208000027744 congestion Diseases 0.000 description 1
- 238000002247 constant time method Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000002934 diuretic Substances 0.000 description 1
- 230000001882 diuretic effect Effects 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 201000006828 endometrial hyperplasia Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- 235000021321 essential mineral Nutrition 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 229940091249 fluoride supplement Drugs 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 229910052732 germanium Inorganic materials 0.000 description 1
- GNPVGFCGXDBREM-UHFFFAOYSA-N germanium atom Chemical compound [Ge] GNPVGFCGXDBREM-UHFFFAOYSA-N 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 229960002449 glycine Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000010015 huanglian Substances 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 235000015243 ice cream Nutrition 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229960005431 ipriflavone Drugs 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 229940004208 lactobacillus bulgaricus Drugs 0.000 description 1
- VMPHSYLJUKZBJJ-UHFFFAOYSA-N lauric acid triglyceride Natural products CCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCC)COC(=O)CCCCCCCCCCC VMPHSYLJUKZBJJ-UHFFFAOYSA-N 0.000 description 1
- 229940069445 licorice extract Drugs 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 235000020640 mackerel Nutrition 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000000845 maltitol Substances 0.000 description 1
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 1
- 235000010449 maltitol Nutrition 0.000 description 1
- 229940035436 maltitol Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 230000003562 morphometric effect Effects 0.000 description 1
- 238000013425 morphometry Methods 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 238000009806 oophorectomy Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000004072 osteoblast differentiation Effects 0.000 description 1
- 230000001599 osteoclastic effect Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 102000002574 p38 Mitogen-Activated Protein Kinases Human genes 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- LCLHHZYHLXDRQG-ZNKJPWOQSA-N pectic acid Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)O[C@H](C(O)=O)[C@@H]1OC1[C@H](O)[C@@H](O)[C@@H](OC2[C@@H]([C@@H](O)[C@@H](O)[C@H](O2)C(O)=O)O)[C@@H](C(O)=O)O1 LCLHHZYHLXDRQG-ZNKJPWOQSA-N 0.000 description 1
- 210000003049 pelvic bone Anatomy 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 235000013550 pizza Nutrition 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000010318 polygalacturonic acid Substances 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001508 potassium citrate Substances 0.000 description 1
- 229960002635 potassium citrate Drugs 0.000 description 1
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 description 1
- 235000011082 potassium citrates Nutrition 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- AAEVYOVXGOFMJO-UHFFFAOYSA-N prometryn Chemical compound CSC1=NC(NC(C)C)=NC(NC(C)C)=N1 AAEVYOVXGOFMJO-UHFFFAOYSA-N 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 235000013580 sausages Nutrition 0.000 description 1
- 208000008742 seborrheic dermatitis Diseases 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 235000011888 snacks Nutrition 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000013222 sprague-dawley male rat Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 235000013616 tea Nutrition 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
Images
Classifications
-
- 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 COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- 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 COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- 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 COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Alternative & Traditional Medicine (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Polymers & Plastics (AREA)
- Pediatric Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physical Education & Sports Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Neurosurgery (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
본 발명은 골질환의 예방 또는 치료에 효과적인 천연물 추출물의 유산균 발효물로서, 더 자세하게는 황련해독탕의 유산균 발효물을 유효성분으로 함유하는 골질환의 예방 또는 치료용 약학적 조성물 및 개선용 건강기능식품 조성물에 관한 것이다. The present invention relates to a fermented product of a lactic acid bacterium of a natural product extract effective for preventing or treating a bone disease, and more particularly, to a pharmaceutical composition for preventing or treating a bone disease containing fermented product of lactic acid bacteria as an active ingredient ≪ / RTI >
Description
본 발명은 황련해독탕의 유산균 발효물을 유효성분으로 함유하는 골질환의 예방 또는 치료용 약학적 조성물 및 건강기능식품에 관한 것이다.
The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating bone diseases containing fermented product of lactic acid bacteria as an active ingredient of Huanghyn Haitang.
정상적인 뼈의 재형성과정은 뼈 형성과 뼈 흡수의 균형으로 이루어지며, 이러한 뼈 형성과 뼈 흡수는 크게 세 가지 세포, 연골세포, 조골세포(osteoblast) 및 파골세포(osteoclast)의 상호작용에 의해 이루어진다. 특히, 조골세포의 증식과 분화는 뼈의 석회화와 밀접한 관계가 있으며 조골세포에서 중요한 전사인자인 RUNX2에 변이가 생기거나 발현량이 감소하면 뼈의 석회화가 일어나지 않는다. RANKL(receptor activator of NF-kB ligand)은 조골세포의 분화과정 동안에 발현되어 파골세포의 분화를 촉진하는 인자로 알려져 있고 OPG(Osteoprotegrin) 역시 조골세포에서 발현되어 RANKL의 역할을 억제함으로써 파골세포의 분화를 억제하는 인자로 알려져 있다.
Normal bone reshaping is a balance of bone formation and bone uptake, and this bone formation and bone uptake is largely due to the interaction of three cells, cartilage cells, osteoblasts and osteoclasts . In particular, the proliferation and differentiation of osteoblasts are closely related to bone calcification, and bone metastases do not occur when mutations or decreased expression levels of RUNX2, an important transcription factor in osteoblast cells, occur. RANKL (receptor activator of NF-kB ligand) is expressed during osteoblast differentiation and promotes osteoclast differentiation. OPG (osteoprotegrin) is also expressed in osteoblasts to inhibit the role of RANKL, Is known as a factor suppressing.
뼈(bone)는 인체의 연조직과 체중을 지탱해주고 내부기관을 둘러싸서 내부 장기를 외부의 충격으로부터 보호한다. 또한 근육이나 장기를 구조적으로 지탱할 뿐만 아니라 체내의 칼슘이나 다른 필수 무기질, 즉 인이나 마그네슘과 같은 물질을 저장하는 인체의 중요한 부분 중 하나이다. 따라서 성장이 끝난 성인의 뼈는 멈추지 않고 죽는 날까지 오래된 뼈는 제거하고 새로운 뼈로 대체하는 생성과 흡수 과정을 매우 역동적, 지속적으로 반복재생 하면서 균형을 유지하게 된다. 이를 골재형성(bone remodeling)이라고 한다. 오래된 뼈는 제거하고 새로운 뼈로 대체하는 뼈의 순환은 성장과 스트레스에 의해서 일어나는 뼈의 미세한 손상을 회복시키고 적절히 뼈의 기능을 유지하는데 필수적이다.
The bone supports the body's soft tissues and body weight and surrounds the internal organs to protect the internal organs from external impact. It is also an important part of the human body that not only supports the muscles or organs but also stores calcium or other essential minerals in the body, such as phosphorus or magnesium. Thus, adult bones do not stop growing until the day they die, and the old bones are removed and replaced with new bones. This is called bone remodeling. The circulation of bones, which remove old bones and replace them with new bones, is essential to restore the fine damage of the bones caused by growth and stress and to maintain proper functioning of the bones.
골재형성에는 크게 두 종류의 세포가 관여하는 것으로 알려져 있다. 두 세포 중 하나는 뼈를 생성하는 조골세포이고, 다른 하나는 뼈를 파괴하는 파골세포이다. 조골세포는 RANKL과 이것의 유도 수용체인 OPG를 생성한다. RANKL이 파골 전구세포 표면에 있는 수용체인 RANK에 결합하면 파골 전구세포가 파골세포로 성숙화되어 골흡수가 일어난다. 그러나 OPG가 RANKL과 결합하면 RANKL과 RANK간 결합이 차단되어 파골세포의 형성이 억제되고 필요 이상의 골 흡수가 일어나지 않게 된다.
It is known that two types of cells are involved in aggregate formation. One of the cells is osteoblast, which produces bone, and the other is osteoclast, which destroys bone. Osteoclasts produce RANKL and its inducible receptor, OPG. When RANKL binds to RANK, a receptor on the surface of osteoclast precursor cells, osteoclast precursor cells mature into osteoclasts and bone resorption occurs. However, when OPG binds to RANKL, the binding between RANKL and RANK is blocked, thereby inhibiting osteoclast formation and preventing bone resorption.
골다공증(osteoporosis)은 여러 가지 원인에 의하여 뼈의 질량이 감소하고 뼈 조직의 미세구조의 퇴화로 골절 위험이 지속적으로 증가하는 질환으로 뼈를 구성하는 미네랄(특히 칼슘)과 기질이 감소한 상태이며, 골재형성의 균형이 깨져서 파골작용이 조골작용보다 증가된 상태에서 발생한다. 정상적인 뼈 내부는 그물망처럼 치밀한 구조를 이루고 있으나, 골다공증의 경우에는 골미세구조 사이의 간격이 넓어지고 미세구조가 얇아져 약해짐으로써 조그만 충격에도 뼈가 쉽게 골절될 위험이 증가하는 질환으로 폐경기 이후 골다공증, 70세 이상의 남녀 노인에게 서서히 발생하며 골반골과 척추뼈의 점진적인 골 손실을 가져오는 노년기 골다공증 및 연령에 상관없이 질병이나 약물, 알코올, 흡연, 사고로 인해 발생하는 2차 골다공증으로 분류된다.
Osteoporosis is a disease in which the bone mass is decreased by various causes and the risk of fracture is continuously increased due to the degeneration of the microstructure of the bone tissue. The bone mineral structure (especially calcium) and the matrix are decreased, The formation of balance is broken and osteoclastic activity occurs in the state of increased osteotomy. Inside the normal bone is dense structure like a net, but in the case of osteoporosis, the gap between the bone microstructures is widened and the microstructure becomes thinner, so that the risk of bone fracture easily increases even after a small impact. It is categorized as osteoporosis in the elderly, which gradually develops in men and women aged 70 years or older, with progressive bone loss in the pelvic bone and vertebrae, and secondary osteoporosis due to disease, drug, alcohol, smoking and accident regardless of age.
골다공증은 현재 가장 중요한 사회적 문제 중 하나로, 미국의 경우 매년 약 26 만명의 여성들에게 유발되고 있으며, 이중 약 12 내지 20% 정도는 사망에 이르고 있다. 사회가 노령화되고 여성들의 사회참여가 활발해지고 있는 상황에서 노인들이나 폐경 후 여성들의 골다공증 및 골다공증으로 인한 골절은 심각한 문제를 야기한다.
Osteoporosis is one of the most important social problems nowadays. In the United States, about 262,000 women are born every year, of which about 12 to 20% die. As society becomes aging and women become more active in society, fractures caused by osteoporosis and osteoporosis of elderly or postmenopausal women cause serious problems.
현재 골다공증 치료제로 사용되고 있는 물질로는 에스트로겐(estrogen), 남성화 스테로이드 호르몬(androgenic anagolic ateroid), 칼슘 제제, 인삼염, 불소 제제, 이프리플라본(Ipriflavone), 비타민 D3 등이 있다. 에스트로겐은 조골세포의 세포고사를 억제하여 세포의 생존기간을 증가시키고 파골세포의 세포고사를 촉진하여 세포의 생존기간을 감소시켜 폐경증상의 치료와 골밀도 유지에 어느 정도 효과적인 방법이나 유방암, 자궁내막 증식증 등을 유발하는 부작용이 있다. 이외에도 파골세포의 활성을 억제하여 골파괴를 억제시키거나 조골세포의 증식을 통해 골재생 단위의 활성을 증가시키는 약물로 칼시토닌, 부갑상선호르몬, 비스포스포네이트 제제 등이 있다. 그러나, 기존 골다공증 치료약제들은 골흡수만을 차단시키거나 골형성을 촉진시키는 효능만을 갖으며 장기간 투여시 많은 부작용을 유발하고 있다. 따라서 장기간 투여에도 지속적인 골밀도 증가 효과를 나타내고 부작용이 적은 안전한 예방 및 치료제 개발이 요구되고 있다.
Currently, estrogen, androgenic anagolic atheroid, calcium, ginseng, fluoride, Ipriflavone, and vitamin D 3 are used in osteoporosis treatment. Estrogen suppresses cell apoptosis of osteoblast cells, thereby increasing the cell survival period. It promotes osteoclast cell apoptosis, thereby decreasing the cell survival period. Thus, estrogen is effective for treatment of menopausal symptoms and for maintaining bone density. Breast cancer, endometrial hyperplasia And the like. In addition, there are calcitonin, parathyroid hormone, and bisphosphonate preparations as a drug that inhibits osteoclast activity and inhibits bone destruction or increases the activity of bone regeneration unit through osteoblast proliferation. However, existing osteoporosis medicines have only the effect of blocking bone resorption or promoting bone formation and causing many side effects in long-term administration. Therefore, it is required to develop a safe preventive and therapeutic agent that exhibits a continuous increase in bone mineral density even after long-term administration and has fewer side effects.
이에, 본 발명자들은 이상과 같은 골다공증과 같은 골질환을 예방 또는 치료하기 위한 천연 약재를 연구하던 중, 황련, 황금, 황백 및 치자의 혼합생약재 추출물의 유산균 발효물이 난소 적출 동물의 골밀도 증가, TRAP 활성 및 다핵성 파골세포 형성 억제 등을 보임으로써 골질환의 예방 또는 치료에 효능이 있음을 확인하고 본 발명을 완성하였다.
Accordingly, the inventors of the present invention have been studying natural medicines for preventing or treating osteoporosis such as osteoporosis as described above. Fermented lactic acid bacteria fermented from mixed medicinal herb extract of Huanglian, golden, Inhibition of osteoclastogenesis, and inhibition of osteoclastogenesis, and inhibition of osteoclast formation, and completed the present invention.
본 발명의 목적은 골질환을 예방 또는 치료할 수 있는 황련해독탕의 유산균 발효물을 유효성분으로 함유하는 약학적 조성물 또는 건강기능식품을 제공하기 위한 것이다.
It is an object of the present invention to provide a pharmaceutical composition or health functional food containing, as an active ingredient, a fermented product of Lactobacillus of Huanghin Haitang, which can prevent or treat bone diseases.
상기의 과제를 해결하기 위해, 황련해독탕의 유산균 발효물을 유효성분으로 포함하는 골질환의 예방 또는 치료용 약학적 조성물을 제공한다.
In order to solve the above-described problems, there is provided a pharmaceutical composition for preventing or treating bone diseases, which comprises a fermented product of Lactobacillus acidus as an active ingredient of Huangrying Haodongtang.
본 발명에서 사용되는 용어 '황련해독탕(黃連解毒湯)'은, 황련(黃連), 황금(黃芩), 황백(黃柏) 및 치자(梔子)로 구성되고, 삼초(상초(上焦), 중초(中焦), 하초(下焦), 즉 사지를 제외한 몸통을 의미함)의 실열(實熱)이 있을 때 효험이 있는 한방유래의 탕약을 의미한다. 열성병(熱性病)으로 염증이 있고 열과 충혈에 의한 정신불안에 출혈이 있으며, 요(尿)가 붉고 심하부(心下部)가 걸려서 저항이 있을 때 처방하고, 또한 하혈, 객혈, 뇌일혈에도 처방하는 것으로 알려져 있다. 최근 연구를 통해 항산화 효과, 항궤양 효과, 이뇨작용, 혈압강하 효과 및 항균 효과 등이 보고된 바 있고, 피부와 관련하여 아토피성 피부염, 접촉성 피부염, 지루성 피부염 등에 효과가 있음이 보고된 바 있다. 본 발명의 황련해독탕은 황련, 황금, 황백 및 치자의 열수추출물인 것이 바람직하다.
As used in the present invention, the term "Hwangryeon detoxified hot water" is composed of Hwangryeong (黄连), 黄 芩 (黄 芩), 黄柏 (黄柏) and Gijang (梔子) It means a herbal medicine derived from a herbal medicine which is efficacious when there is a heat of., Which means 중), 초), 焦,, the body excluding the limbs. It is prescribed when there is inflammation with fever disease (fever disease), mental anxiety caused by fever and congestion, bleeding in urine, redness in the lower part of the heart (lower part of the heart) and resistance, and also prescribed in hemorrhage, hemoptysis, . Recent studies have reported antioxidative, anti-ulcer, diuretic, hypotensive and antimicrobial effects, and have been reported to be effective in atopic, dermatitis and seborrheic dermatitis associated with the skin . It is preferred that the phytophthora capsicum of the present invention is a hot-water extract of phloem, golden, yellow-white and ginger.
본 발명에서 사용되는 용어 '유산균 발효물'은, 유산균이 성장할 수 있는 배지에 유산균을 접종하고 배양한 배양물로부터 수득한 배양결과물을 의미한다. 본 발명의 유산균 발효물은 상기 황련해독탕에 유산균을 접종하고, 호기성 대기하에, 35℃ 내지 40℃의 온도 및 1일 내지 3일의 시간 동안 발효시킨 것이 바람직하다.
The term " fermented product of lactic acid bacteria " used in the present invention means the result of culture obtained from a culture obtained by inoculating lactic acid bacterium into a medium in which lactic acid bacteria can grow. The fermented product of lactic acid bacteria of the present invention is preferably fermented at a temperature of 35 ° C to 40 ° C and for a period of 1 day to 3 days under an aerobic atmosphere by inoculating the fermented soybean flour with the fermented soybean flour.
상기 유산균은 락토바실루스(Lactobacillus) 속 또는 비피도박테리움(Bifidobacterium) 속인 것이 바람직하다.
The lactic acid bacterium is preferably in the genus Lactobacillus or Bifidobacterium.
상기 락토바실루스(Lactobacillus) 속은, 락토바실루스 카제이(Lactobacillus casei), 락토바실루스 아시도필루스(Lactobacillus acidophilus), 락토바실루스 플란타룸(Lactobacillus plantarum), 락토바실루스 퍼멘텀(Lactobacillus fermentum), 락토바실루스 커바투스(Lactobacillus curvatus), 락토바실루스 콘푸수스(Lactobacillus confusus) 및 락토바실루스 가세리(Lactobacillus gasseri) 균주로 구성된 군으로부터 선택되는 1종 이상인 것이 바람직하다.
The Lactobacillus genus includes Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus fermentum, Lactobacillus spp., Lactobacillus spp. It is preferably at least one selected from the group consisting of Lactobacillus curvatus, Lactobacillus confusus, and Lactobacillus gasseri.
상기 비피도박테리움(Bifidobacterium) 속은, 비피도박테리움 브레브(Bifidobacterium breve) 또는 비피도박테리움 써모필럼(Bifidobacterium thermophilum) 균주인 것이 바람직하다.
It is preferable that the genus Bifidobacterium is Bifidobacterium breve or Bifidobacterium thermophilum.
본 발명에서 사용되는 용어 '골질환'은, 골밀도가 감소되어 일어나는 모든 질환을 의미하는 것으로, 골다공증, 골형성 부전증, 치주질환 또는 골절 등을 포함한다.
The term 'bone disease' as used in the present invention means all diseases caused by a decrease in bone density, and includes osteoporosis, osteoporosis, periodontal disease, or fracture.
본 발명에서는 황련해독탕의 유산균 발효물을 함유하는 조성물이 골질환의 예방 또는 치료에 효과가 있음을 확인하였다. 본 발명의 일 실시예에 따르면, 골질환의 지표가 되는 파골세포 분화마커인 TRAP 활성 및 다핵성 파골세포 형성 억제효과를 확인함으로써, 골질환의 예방 또는 치료 효능이 있음을 확인하였다.
In the present invention, it was confirmed that a composition containing a fermented product of Lactobacillus acidophilus of Rhodiola is effective for preventing or treating bone diseases. According to one embodiment of the present invention, TRAP activity, which is an osteoclast differentiation marker which is an index of bone disease, and inhibitory effect on formation of polynuclear osteoclast were confirmed, and it was confirmed that osteoporosis is prevented or treated.
본 발명에서 사용되는 용어 '예방'은, 상기 황련해독탕의 유산균 발효물을 함유하는 조성물의 투여로 질환을 억제 또는 지연시키는 모든 행위를 의미한다. 또한, 본 발명에서 사용되는 용어 '치료'는, 상기 황련해독탕의 유산균 발효물을 함유하는 조성물의 투여로 질환의 증세가 호전되거나 완치되는 모든 행위를 의미한다.
The term " prevention " as used in the present invention means any action that inhibits or delays the disease by administration of a composition containing the fermented product of the lactic acid bacteria of the phytophthora infestans. The term " treatment " used in the present invention means all the actions of improving or ameliorating the symptoms of the disease by administration of the composition containing the fermented product of the lactic acid bacteria of the phytophthora infestans.
본 발명의 조성물은 투여를 위하여, 상기 기재한 유효성분 이외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 포함할 수 있다. 상기 담체, 보형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.
The composition of the present invention may contain, for administration, a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described effective ingredients. The carrier, the beating agent and the diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose , Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
본 발명의 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사 용액의 형태로 제형화하여 사용할 수 있다. 상세하게는, 제형화할 경우 통상 사용하는 충진제, 중량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제로는 정제, 환제, 산제, 과립제, 캡슐제 등을 포함하나, 이에 한정되는 것은 아니다. 이러한 고형제제는 상기 황련해독탕의 유산균 발효물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘 카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등을 첨가하여 조제될 수 있다. 비경구 투여를 위한 제제는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제 및 과제를 포함한다. 비수성 용제 및 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 오일, 에틸올레이트와 같은 주사가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로솔, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.
The composition of the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository or sterilized injection solution according to a conventional method. In detail, when formulating, it can be prepared by using diluents or excipients such as fillers, weighing agents, binders, humectants, disintegrants, surfactants and the like which are generally used. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like in the fermented product of the lactic acid bacteria of the phytophthora infestans. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration, liquid paraffin, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and tasks. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. As a base for suppositories, it is possible to use witepsol, macrosole, tween 61, cacao paper, laurin, glycerogelatin and the like.
본 발명의 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여 경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 상기 황련해독탕의 유산균 발효물의 일일 투여량은 바람직하게는 1 mg/kg 내지 500 mg/kg이며, 필요에 따라 일일 1회 내지 수회로 나누어 투여할 수 있다.
The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dose may be determined depending on the condition and weight of the patient, The mode of administration, the route of administration, and the time, but may be suitably selected by those skilled in the art. The daily dose of the fermented lactic acid bacteria of the Huangliao powder is preferably 1 mg / kg to 500 mg / kg, and may be administered once to several times per day, if necessary.
또한, 본 발명은 황련해독탕의 유산균 발효물을 유효성분으로 함유하는 골질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다.
The present invention also provides a health functional food composition for preventing or ameliorating osteoporosis, which contains fermented lactic acid bacteria as an active ingredient of Huangryinghaotang.
상기 건강기능식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 천연 과일 주스 및 과일 주스 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 또한, 건강기능식품은 육류, 소세지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 껌류, 아이스크림류, 스프, 음료수, 차, 기능수, 드링크제, 알콜 및 비타민 복합제 중 어느 하나의 형태일 수 있다.
The health functional food may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. It can also contain natural fruit juices and pulp for the production of fruit juices and vegetable drinks. These components may be used independently or in combination. The health functional food may be in the form of any one of meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, .
또한 상기 건강기능식품은 식품첨가물을 추가로 포함할 수 있으며, "식품첨가물"로서의 적합여부는 다른 규정이 없는 한 식품의약품안정청에 승인된 식품첨가물공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.
In addition, the health functional food may further include food additives, and the suitability of the food functional food as a "food additive" Standards and standards.
상기 "식품첨가물공전"에 수재된 품목으로 예를 들어, 케톤류, 글리신, 구연산 칼륨, 니코틴산, 계피산 등의 화학적 합성품, 감색소, 감초추출물, 결정셀룰로오스, 고랭색소, 구아검 등의 천연첨가물, L-글루타민산나트륨제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합 제제류 등을 들 수 있다.
Examples of the products that have been used in the above-mentioned "food additives" include natural products such as ketones, chemical products such as glycine, potassium citrate, nicotinic acid and cinnamic acid, sensory coloring matter, licorice extract, crystalline cellulose, high- - Mixed preparations such as a sodium glutamate preparation, a noodle-added alkaline agent, a preservative preparation, a tar coloring agent and the like.
이때, 건강기능식품을 제조하는 과정에서 음료를 포함한 식품에 첨가되는 본 발명에 따른 황련해독탕의 유산균 발효물은 필요에 따라 그 함량을 적절히 가감할 수 있으며, 바람직하게는 식품 100 중량%에 1 내지 15 중량% 포함되도록 첨가하는 것이 바람직하다.
At this time, the fermented product of Lactobacillus acidophilus according to the present invention, which is added to foods containing beverages in the process of manufacturing a health functional food, can be appropriately added or decreased as needed, and preferably 100% By weight to 15% by weight.
본 발명은 골질환의 예방 또는 치료에 효과적인 황련해독탕의 유산균 발효물로서, 골질환의 예방 또는 치료 조성물로 약학적으로 이용 가능할 뿐 아니라 건강기능식품으로서도 유용하게 이용될 수 있다.
The present invention is a fermented product of Lactobacillus acidus which is effective for preventing or treating bone diseases, and can be used not only as a pharmaceutical composition for preventing or treating bone diseases, but also as a health functional food.
도 1은, 본 발명의 일 실시예에 따른 RAW264.7 세포에서 황련해독탕(HRT) 및 황련해독탕 발효물(fHRT)의 TRAP 활성에 대한 효과를 나타낸 그래프이다.
도 2는, 본 발명의 일 실시예에 따른 황련해독탕(HRT) 및 황련해독탕 발효물(fHRT)의 (A) TRAP 활성 억제 효능, (B) TRAP 양성 다핵성파골세포 형성 억제 효능 및 (C) TRAP 양성 다핵성파골세포 형성 억제 효과를 확인한 세포 사진에 대한 결과를 나타낸 것이다.
도 3은, 본 발명의 일 실시예에 따른 황련해독탕(HRT) 및 황련해독탕 발효물(fHRT)의 (A) 에틸아세테이트(EA) 분획물의 TRAP 활성 억제 효능, (B) 부탄올(BU) 분획물의 TRAP 활성 억제 효능, (C) 부탄올(BU) 분획물의 TRAP 양성 다핵성파골세포 형성 억제 효능 및 (D) TRAP 양성 다핵성파골세포 형성 억제 효과를 확인한 세포 사진에 대한 결과를 나타낸 것이다.
도 4는, 본 발명의 일 실시예에 따른 공생배양 조건에서의 황련해독탕(HRT) 및 황련해독탕 발효물(fHRT)의 부탄올 분획물의 (A) TRAP 활성 억제 효능, (B) TRAP 양성 다핵성파골세포 형성 억제 효능 및 (C) TRAP 양성 다핵성파골세포 형성 억제 효과를 확인한 세포 사진에 대한 결과를 나타낸 것이다.
도 5는, 본 발명의 일 실시예에 따른 (A) 황련해독탕(HRT) 및 황련해독탕 발효물(fHRT), (B) 황련해독탕(HRT) 및 황련해독탕 발효물(fHRT)의 에틸아세테이트 분획물(EA) 및 (C) 황련해독탕(HRT) 및 황련해독탕 발효물(fHRT)의 부탄올 분획물(BU)의 세포 생장에 대한 효과를 나타낸 그래프이다.
도 6은, 본 발명의 일 실시예에 따른 (A) 황련해독탕 부탄올 분획(HRT-BU) 및 황련해독탕 발효물 127 부탄올 분획(fHRT-BU127) 및 (B) 황련해독탕 부탄올 분획(HRT-BU) 및 황련해독탕 발효물 166 부탄올 분획(fHRT-BU166)의 MAPK 및 NF-kB 신호전달계에 대한 효과를 나타낸 것이다.
도 7은, 본 발명의 일 실시예에 따른 (A) 황련해독탕 부탄올 분획(HRT-BU) 및 황련해독탕 발효물 127 부탄올 분획(fHRT-BU127) 및 (B) 황련해독탕 부탄올 분획(HRT-BU) 및 황련해독탕 발효물 166 부탄올 분획(fHRT-BU166)의 NFATc1 및 Atp6v0d2 단백질 발현에 대한 효과를 나타낸 것이다.
도 8은, 본 발명의 일 실시예에 따른 황련해독탕 부탄올 분획(HRT-BU), 황련해독탕 발효물 127 부탄올 분획(fHRT-BU127) 및 황련해독탕 발효물 166 부탄올 분획(fHRT-BU166)의 NF-kB에 의해 조절되는 유전자 (A) ICAM-1, (B) NFkb2, (C) TNF-α 및 (D) I-kBα의 mRNA 발현에 대한 QPCR 결과를 나타낸 것이다.
도 9는, 본 발명의 일 실시예에 따른 황련해독탕 부탄올 분획(HRT-BU), 황련해독탕 발효물 127 부탄올 분획(fHRT-BU127) 및 황련해독탕 발효물 166 부탄올 분획(fHRT-BU166)의 NFATc1을 과발현시킨 BMM세포에 대한 (A) TRAP 활성 억제 효능, (B) TRAP 양성 다핵성파골세포 형성 억제 효능 및 (C) Western blot 결과를 나타낸 것이다.
도 10은, 본 발명의 일 실시예에 따른 난소 절제 랫트의 (A) bone mineral density(BMD), (B) bone volume/trabecular volume(BV/TV), (C) bone surface/bone volume(BS/BV), (D) Trabecular Thinckness(Tb.Th), (E) Trabecular separation(Tb.Sp) 및 (F) Trabecular number(Tb.N)에 대한 결과를 나타낸 것이다.
도 11은, 본 발명의 일 실시예에 따른 난소 절제 랫트의 대퇴골 Micro-CT 결과를 나타낸 것이다. FIG. 1 is a graph showing the effect of TRH activity on the HRT and fHRT in RAW264.7 cells according to an embodiment of the present invention.
FIG. 2 is a graph showing the effect of (A) inhibiting TRAP activity, (B) inhibiting the formation of TRAP-positive polynuclear osteoclasts, and (f) C) results of cell photographs confirming the inhibitory effect of TRAP-positive polynuclear osteoclast formation.
FIG. 3 is a graph showing the effect of (A) ethyl acetate (EA) fraction on the TRAP activity inhibitory effect of (B) butanol (BU) on the HRT and the fHRT according to an embodiment of the present invention, (D) TRAP-positive polynuclear osteoclast formation inhibitory effect of (C) butanol (BU) fractions of TRAP-positive polynuclear osteoclast formation inhibitory effect of the fractions.
FIG. 4 is a graph showing the effect of (A) inhibiting TRAP activity of the butanol fraction of the fermented soybean oil (HRT) and the fermented soybean fermented product (fHRT) under the symbiotic culture conditions according to an embodiment of the present invention, (B) (C) TRAP-positive polynuclear osteoclast formation inhibitory effect on osteoclast-like cells.
FIG. 5 is a graph showing the results of (A) HRT and HRT, (b) HRT and HRT, and (BU) of the ethyl acetate fraction (EA) and (C) the Huangliai detoxified (HRT) and the Huangliao detoxified fermented product (fHRT).
(HRT-BU) and 127-butanol fraction (fHRT-BU127) of fermented seaweed fermented product (B), and (B) HRT -BU) and the 166-butanol fraction (fHRT-BU166) of the fermented soybean milk fermented product on the MAPK and NF-kB signaling system.
(HRT-BU) and 127-butanol fraction (fHRT-BU127) of fermented seaweed fermented product (B), and (B) HRT -BU) and the 166-butanol fraction (fHRT-BU166) of the Huangliao and Taotang fermented products were tested for NFATc1 and Atp6v0d2 protein expression.
(FHRT-BU127) and the 166-butanol fraction (fHRT-BU166) of the fermented seaweed fermented product according to one embodiment of the present invention, HRT- (A) ICAM-1, (B) NFkb2, (C) TNF-? And (D) I-kB?
(FHRT-BU127) and the 166-butanol fraction (fHRT-BU166) of the fermented seaweed fermented product according to an embodiment of the present invention, (A) TRAP activity inhibitory effect on BMM cells overexpressing NFATc1, (B) TRAP-positive polynuclear osteoclast formation inhibitory effect, and (C) Western blotting results.
FIG. 10 is a graph showing bone mineral density (BMD), bone volume / trabecular volume (BV / TV), bone surface / bone volume (BS) of ovariectomized rats 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).
11 is a micro-CT result of a femur of an ovariectomized rat according to an embodiment of the present invention.
이하, 하기 제조예 및 실시예에 의하여 본 발명을 더욱 상세하게 설명하고자 한다. 단, 하기 제조예 및 실시예는 본 발명을 예시하기 위한 것일 뿐 본 발명의 범위가 이들만으로 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to the following Production Examples and Examples. However, the following Preparation Examples and Examples are for illustrating the present invention, but the scope of the present invention is not limited thereto.
실시예Example
: :
황련해독탕Hwang Ryun Ha
유산균 Lactobacillus
발효물Fermentation product
((
fHRTfHRT
) 제조) Produce
1) HRT(황련해독탕) 제조1) Manufacture of HRT
황련, 황금, 황백 및 치자를 각 250 g씩 준비하여 총 1000 g을 10 ℓ 생수에 넣은 후 1시간 침적하였다. 그리고 115℃에서 180분간 열탕 추출하였다. 추출물은 test sieve(106 ㎛)를 이용하여 여과하여 황련해독탕을 얻었다.
250 g each of golden, yellow, white, and germanium were prepared, and a total of 1000 g was placed in 10 L water and immersed for 1 hour. Then, the hot water was extracted at 115 ° C for 180 minutes. The extract was filtered using a test sieve (106 ㎛) to obtain a potash.
2) 황련해독탕의 유산균 발효2) Fermentation of Lactobacillus acidophore
fHRT(황련해독탕 유산균 발효물)을 제조하기 위해 락토바실루스(Lactobacillus) 속 및 비피도박테리움(Bifidobacterium) 속을 한국식품연구원(KFRI)으로부터 받아서 사용하였으며, 하기 표 1에 황련해독탕 유산균 발표물을 제조하기 위해 사용한 발효균주를 나타내었다. MRS 배지에서 37℃로 24시간 동안 2회 계대배양 한 후 배양액을 다시 배지에 주입하였다. 그리고 1×106 내지 5×106 CFU/ml의 개체군을 얻기 위하여 적당히 희석한 후 접종원으로 사용하였다. 발효를 위하여, 상기 실시예 1)에서 제조한 HRT(황련해독탕) 5 ml를 상기 접종원 0.05 ml와 함께 캡이 있는 시험관에 주입하였다. 그리고 48시간 동안 37℃를 유지하면서 배양하였다. Lactobacillus genus and Bifidobacterium genus were purchased from Korea Food Research Institute (KFRI) for the production of fHRT (fermented product of phytophthora infestans Lactobacillus acidophilus) The fermentation broth used for the preparation of the fermentation broth was shown. After subculturing in MRS medium for 2 hours at 37 ° C, the culture medium was again injected into the medium. And was appropriately diluted to obtain a population of 1 × 10 6 to 5 × 10 6 CFU / ml and used as an inoculation source. For fermentation, 5 ml of the HRT (Huangryinghaeganggang) prepared in Example 1) was injected into a cap test tube together with 0.05 ml of the inoculum. And cultured at 37 DEG C for 48 hours.
*DSM: Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH, Braunschweig, Germany
*NCFB: National Collection of Food Bacteria, Reading United Kingdom
*IFO: Instite for Fermentation, Osaka, Japan* 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: Instite for Fermentation, Osaka, Japan
실험예Experimental Example
1: One:
inin
--
vitrovitro
분석analysis
1) TRAP 활성 분석1) TRAP activity analysis
파골세포 분화마커인 TRAP 효소 활성을 억제하는 효능을 확인하기 위하여, RANKL에 의해 파골 세포로 분화하는 RAW264.7 세포에 HRT(황련해독탕) 및 fHRT(황련해독탕 발효물)을 처리한 후 TRAP 활성을 측정하였다. 파골세포는 마우스의 골수세포로부터 얻었다. 골수세포는 M-CSF(60 ng/ml)를 함유한 10% FBS로 이뤄진 α-MEM에서 3일 동안 배양하여 골수 세포 유래 대식세포(BMMs)를 수득하였으며, 이를 파골세포 전구 세포로 사용하였다. 파골세포 생성을 위해, BMMs를 M-CSF(60 ng/ml) 및 RANKL(150 ng/ml)와 4일 동안 배양하였다. 조골전구세포 및 골수세포를 포함하는 공생배양으로부터 파골세포 생성을 위해, 조골전구세포는 신생마우스의 두개골로부터 준비하였다. 골수세포(BMCs) 및 조골전구세포는 1.25(OH)2D3(1×10-8 M)의 존재하에 6일 동안 공생배양하였다. 배양된 세포는 TRAP 활성 측정을 위해 고정하고 착색하였다. TRAP 활성은 기질(q-나이트로페닐포스페이트, q-nitrophenyl phosphate)을 처리한 후 405 nm 흡광도에서 측정하였다.
In order to confirm the effect of inhibiting the activity of TRAP enzyme, which is an osteoclast differentiation marker, RAW264.7 cells differentiated into osteoclasts by RANKL were treated with HRT (fungicide) and fHRT Activity was measured. Osteoclasts were obtained from mouse bone marrow cells. Bone marrow cells were cultured in α-MEM containing 10% FBS containing M-CSF (60 ng / ml) for 3 days to obtain bone marrow-derived macrophages (BMMs), which were used as osteoclast precursor cells. For osteoclastogenesis, BMMs were cultured with M-CSF (60 ng / ml) and RANKL (150 ng / ml) for 4 days. For osteoclastogenesis from syngeneic cultures containing osteoprogenitor cells and bone marrow cells, osteoprogenitor cells were prepared from the skull of neonatal mice. Bone marrow cells (BMCs) and osteoblast precursors were co-cultured for 6 days in the presence of 1.25 (OH) 2 D 3 (1 × 10 -8 M). The cultured cells were fixed and stained for TRAP activity measurement. TRAP activity was measured at 405 nm absorbance after treatment with substrate (q-nitrophenyl phosphate).
① HRT(황련해독탕) 및 fHRT(황련해독탕 발효물)의 TRAP 효소 억제 활성① TRAP enzyme inhibitory activity of HRT (Huangliao Yangtang) and fHRT
상기 실시예에서 제조된 황련해독탕(HRT) 및 황련해독탕 발효물(fHRT) 각각 100 ㎍/ml를 RAW264.7 세포에 처리하여 TRAP 활성 억제 효과를 분석하였으며, 도 1에 결과를 나타내었다. 도 1에 나타난 바와 같이, HRT와 비교하여 fHRT127은 13%, fHRT144는 6% 그리고 fHRT166은 8%로 TRAP 활성을 감소시키는 것을 확인하였다.
The effect of inhibiting TRAP activity was analyzed by treating RAW264.7 cells with 100 ㎍ / ml of each of HRT and fHRT prepared in the above examples, and the results are shown in Fig. As shown in Fig. 1, it was confirmed that fHRT127, fHRT144 and fHRT166 decreased TRAP activity by 13%, fHRT144 and fHRT166, respectively, as compared with HRT.
② HRT(황련해독탕) 및 fHRT(황련해독탕 발효물)의 농도별 TRAP 효소 억제 활성② TRAP enzyme inhibitory activity by concentration of HRT (Huangliao Yangtang) and fHRT
농도별 효과를 알아보기 위하여, fHRT127 및 fHRT166과 HRT를 12.5 내지 200 ㎍/ml 농도로 처리하여 TRAP 활성 억제 효과 및 TRAP 양성 다핵성 파골세포 형성에 미치는 효과를 분석하였으며, 도 2에 나타내었다. 도 2에 나타난 바와 같이, 50, 100 및 200 ㎍/ml HRT는 TRAP 효소 활성을 19, 67 및 94% 억제하였으며, 100 ㎍/ml에서 HRT와 비교하여 fHRT127은 11%, fHRT166은 18%로 추가적인 TRAP 활성 억제효능을 나타내었다. 또한, 100 ㎍/ml에서 HRT 및 두 종의 fHRT가 약 90%의 TRAP 양성 다핵성 파골세포 형성 억제 효능을 보이는 것을 확인하였다.
In order to investigate the effect of concentrations, the effects of treating fHRT127 and fHRT166 and HRT at a concentration of 12.5 to 200 / / ml on TRAP inhibitory effect and TRAP positive polynuclear osteoclast formation were analyzed and shown in Fig. As shown in Figure 2, 50, 100, and 200 / / ml HRT inhibited TRAP enzyme activity by 19, 67, and 94%, and compared with HRT at 100 / / ml, fHRT127 was 11% and fHRT166 was 18% TRAP inhibitory activity. In addition, HRT and HRT of 100 μg / ml and fHRT of two species showed about 90% inhibition of TRAP positive polynuclear osteoclast formation.
③ HRT(황련해독탕) 및 fHRT(황련해독탕 발효물) 분획물의 TRAP 효소 억제 활성 및 TRAP 양성 다핵성 파골세포 생성 억제 활성 분석③ Analysis of TRAP enzyme inhibitory activity and TRAP-positive polynuclear osteoclastogenesis inhibitory activity of the HRT (fungicide) and fHRT
상기 실시예에서 제조된 HRT 또는 fHRT을 에틸아세테이트(EA) 및 n-부탄올(BU)에 계대 용매 추출을 사용하여 분별하였으며, 각 분획은 건조시킨 후 -20℃ 건조기에 보관하였다. 각 분획물을 0 내지 30 ㎍/ml로 처리한 후 TRAP 효소 억제 활성을 분석하였으며, 그 결과를 도 3에 나타내었다. HRT or fHRT prepared in the above example was fractionated by fractional solvent extraction in ethyl acetate (EA) and n-butanol (BU), and each fraction was dried and stored in a -20 ° C dryer. Each fraction was treated with 0 to 30 占 퐂 / ml and the TRAP enzyme inhibitory activity was analyzed. The results are shown in Fig.
도 3에 나타난 바와 같이, 10 ㎍/ml 농도에서 HRT-EA(황련해독탕 에틸아세테이트 분획)와 비교하여 fHRT-EA127(황련해독탕 발효물 127의 에틸아세테이트 분획)은 24%, fHRT-EA166(황련해독탕 발효물 166의 에틸아세테이트 분획)은 9%의 TRAP 활성 억제 효능을 나타내었다. 또한, 0 ㎍/ml 농도와 비교하여, 3, 10, 30 ㎍/ml 농도에서 HRT-BU(황련해독탕 부탄올 분획)은 각각 17%, 67% 및 95%의 TRAP 억제 활성을 보였으며, 3 ㎍/ml HRT-BU와 비교하여 fHRT-BU127(황련해독탕 발효물 127의 부탄올 분획)은 23%, fHRT-BU166(황련해독탕 발효물 166의 부탄올 분획)은 32%로 추가적인 TRAP 억제 효능을 보였다. 10 ㎍/ml HRT-BU와 비교해서는 fHRT-BU127은 21%, fHRT-BU166은 22%의 추가적인 억제효능을 보였으며, 30 ㎍/ml HRT-BU와 비교하여 fHRT-BU127은 3% 그리고 fHRT-BU166은 3%의 추가적인 TRAP 활성 억제 효능을 나타내었다.As shown in FIG. 3, fHRT-EA127 (ethylacetate fraction of Huangliao detoxified fermented product 127) was 24% and fHRT-EA166 (ethyl acetate fraction) was 10% The ethylacetate fraction of 166 fermented broth) showed 9% TRAP inhibitory activity. HRT-BU (17%, 67% and 95%, respectively) showed TRAP inhibitory activity at concentrations of 3, 10 and 30 ㎍ / ml compared with 0 ㎍ / 23% of fHRT-BU127 (butanol fraction of Huangliao detoxified fermented product 127) and 32% of fHRT-BU166 (butanol fraction of Huangliao detoxified fermented product 166) have an additional TRAP inhibitory effect compared to ㎍ / ml HRT-BU It looked. Compared to 10 ㎍ / ml HRT-BU, fHRT-BU127 showed 21% inhibition and fHRT-BU166 had 22% inhibitory effect. Compared with 30 ㎍ / ml HRT-BU, fHRT- BU166 showed an additional TRAP inhibitory effect of 3%.
또한, 0 ㎍/ml와 비교하여 3, 10, 30 ㎍/ml HRT-BU에서는 각각 55%, 97% 및 98%의 TRAP 양성 다핵성 파골세포 형성 억제 효능을 보였으며, 3 ㎍/ml HRT-BU와 비교하여 fHRT-BU127 및 fHRT-BU166은 34%의 추가적인 TRAP 양성 다핵성 파골세포 형성 억제 효능을 나타내었다.
In addition, 3, 10, and 30 ㎍ / ml HRT-BU showed 55%, 97%, and 98% inhibitory effects on TRAP-positive polynuclear osteoclast formation compared with 0 ㎍ / Compared with BU, fHRT-BU127 and fHRT-BU166 showed 34% additional TRAP-positive polynuclear osteoclast formation inhibitory activity.
④ 공생배양 조건에서 HRT-BU 및 fHRT-BU의 TRAP 활성 억제 및 TRAP 양성 다핵성 파골세포 형성 억제 효과 ④ Inhibition of TRAP activity and inhibition of TRAP-positive polynuclear osteoclast formation in HRT-BU and fHRT-BU in the symbiotic culture condition
HRT-BU 및 fHRT-BU의 골수세포와 조골전구세포(pre-osteeoblast) 공생배양 조건에서 TRAP 효소 활성 및 TRAP 양성 다핵성 파골세포 형성에 미치는 영향을 확인하기 위하여, 상기 HRT-BU 및 fHRT-BU를 0 내지 30 ㎍/ml 처리하여 분석하였으며, 결과를 도 4에 나타내었다. 도 4에 나타난 바와 같이, 0 ㎍/ml와 비교하여 10 및 30 ㎍/ml의 HRT-BU는 53 및 95%의 TRAP 활성 억제 효능을 나타냈으며, 10 ㎍/ml HRT-BU와 비교하여 fHRT-BU127은 24%, fHRT-BU166은 33%의 추가적인 억제활성을 보였다. 또한, 30 ㎍/ml HRT-BU와 비교하여 fHRT-BU127 및 fHRT-BU166은 각각 3%의 추가적인 TRAP 활성 억제 효능을 나타내었다.BU and fHRT-BU in order to confirm the effect of TRAP enzyme activity and TRAP-positive polynuclear osteoclast formation on the bone marrow cells of HRT-BU and fHRT-BU and pre-osteoblast co- Was treated at 0 to 30 占 퐂 / ml and analyzed. The results are shown in Fig. As shown in FIG. 4, HRT-BU of 10 and 30 / / ml showed inhibitory effects on TRAP activity of 53 and 95%, compared with 0 / / ml, and fHRT- The inhibitory activity of BU127 was 24% and that of fHRT-BU166 was 33%. In addition, fHRT-BU127 and fHRT-BU166 showed an additional 3% inhibition of TRAP activity compared with 30 ㎍ / ml HRT-BU.
또한, 0 ㎍/ml와 비교하여 3, 10, 30 ㎍/ml HRT-BU는 34%, 96% 및 98%의 TRAP 양성 다핵성 파골세포 형성 억제 효능을 보였으며, 3 ㎍/ml HRT-BU와 비교하여 fHRT-BU127 및 fHRT-BU166은 각각 34%의 추가적인 TRAP 양성 다핵성 파골세포 형성 억제 효능을 나타내었다. 10 ㎍/ml HRT-BU와 비교한 경우에는 각각 3%의 추가적인 TRAP 양성 다핵성 파골세포 형성 억제 효능을 보였다.
In addition, 3, 10 and 30 ㎍ / ml HRT-BU showed 34%, 96% and 98% inhibition of TRAP positive polynuclear osteoclast formation compared to 0 ㎍ / ml and 3 ㎍ / ml HRT-BU , FHRT-BU127 and fHRT-BU166 inhibited 34% of additional TRAP-positive polynuclear osteoclast formation, respectively. Compared with 10 ㎍ / ml HRT-BU, each showed 3% additional TRAP-positive polynuclear osteoclast formation inhibitory activity.
2) 세포 생장 분석(cell viability assay)2) Cell viability assay (cell viability assay)
BMM 세포(1×104 cell/well)는 M-CSF(60 ng/ml) 존재하에 96-well plate에서 3일 동안 다양한 농도(12.5 내지 200 ㎍/ml)로 시료와 함께 배양하였다. Cell counting Kit-8(CCK-8)은 세포 생존능 측정에 사용하였으며, 데이터는 3번 반복실험의 평균과 표준오차로 나타내었다. 도 5에 나타난 바와 같이, 실험에 사용한 HRT(황련해독탕), fHRT(황련해독탕 발효물)이 생장을 억제하지 않음을 확인하였다. 또한, 10 ㎍/ml 농도에서 HRT-EA(황련해독탕 에탄올아세테이트 분획)은 세포 생장을 억제하지 않았으나, fHRT-EA127(황련해독탕 발효물 127의 에틸아세테이트 분획)은 26%, fHRT-EA166(황련해독탕 발효물 166의 에틸아세테이트 분획)은 22%의 추가적인 세포생장 억제를 하였으며, 30 ㎍/ml HRT-EA와 비교하여 fHRT-EA127은 35%, fHRT-EA166은 36%의 추가적인 세포생장을 억제하였다. 반면, fHRT-BU(황련해독탕 발효물의 부탄올 분획)은 세포생장 억제 활성이 나타나지 않았다.
BMM cells (1 × 10 4 cells / well) were cultured in 96-well plates in the presence of M-CSF (60 ng / ml) for 3 days at various concentrations (12.5-200 ㎍ / ml). Cell counting Kit-8 (CCK-8) was used to measure cell viability. The data were expressed as the mean and standard error of three replicate experiments. As shown in Fig. 5, it was confirmed that the HRT (Huangliao detoxification) and fHRT (Huangliao detoxified fermentation product) used in the experiment did not inhibit the growth. HRT-EA did not inhibit cell growth, but fHRT-EA127 (ethylacetate fraction of Rhodiola fermented product 127) was 26% and fHRT-EA166 And the fHRT-EA166 had an additional cell growth of 36% compared with 30 ㎍ / ml HRT-EA. The fHRT-EA127 and the fHRT-EA166 showed an additional cell growth of 22% Respectively. On the other hand, fHRT-BU (butanol fraction of Huangliao mackerel fermented product) showed no cell growth inhibitory activity.
따라서, 상기 실험예 1의 1) 및 2)의 결과에 따라, 세포 독성이 없으면서 추가적인 TRAP 활성 억제 효능을 갖는 HRT-BU 및 fHRT-BU 분획을 다른 in-vitro 실험에 사용하였다.
Therefore, HRT-BU and fHRT-BU fractions with no cytotoxicity and with additional TRAP inhibitory activity were used for other in-vitro experiments according to the results of 1) and 2) of Experimental Example 1 above.
3) Western blot 분석3) Western blot analysis
BMMs는 차가운 인산완충식염수(PBS)로 세척하고 50 mM tris-염산(pH 8.0), 5 mM EDTA, 150 mM 염화나트륨, 1% NP-40, 0.1% SDS, 1 nM PMSF, 분해효소 저해제 혼합 타블렛 및 인산화효소 저해제 혼합 타블렛으로 구성된 단백질 추출 완충용액(proteinn extraction buffer)에 녹였다. 세포 분해물은 4℃에서 10,000 ×g으로 15분 동안 원심분리하였다. 단백질 농도는 BCA 단백질 분석 키트를 사용하여 측정하였으며, 단백질 시료(20 ㎍)은 시료 완충액(100 nM tris-염산, 2% SDS, 1% 2-메캅토에탄올, 2% 글리세롤 및 0.01% 브로모페놀 블루, pH 7.6)과 혼합하고 5분 동안 95℃에서 배양하고 12% 폴리아크릴아민 겔에 점적하였다. 전기영동법은 mini protean 3 cell(Bio-Rad, Hercules, CA, USA)를 사용하여 수행하였으며, 겔 위에 분리된 단백질은 PVDF 막으로 옮겼다. 막은 blocking 완충액(10 mM tris-염산(pH 7.5), 150 mM 염화나트륨, 0.1% Tween 20 및 3% non-fat dry milk) 내에서 배양한 후, 1:1000으로 희석된 1차 항체와 함께 상온에서 2시간 동안 배양하였다. 세척 완충액(10 mM tris-염산(pH 7.5), 150 mM 염화나트륨 및 0.1% Tween 20)으로 각 10분 동안 3차례 세척한 후, 1:2000으로 희석된 2 차 항체(Cell Signaling Technology Inc., Danvers, MA, USA)와 1시간 동안 배양하였다. 그 후, 막을 세척 완충액(10 nM tris-염산(pH 7.5), 150 mM 염화나트륨 및 0.1% Tween 20)으로 각 10분 동안 3차례 세척한 후 SuperSignal West Femto Maximum Sensitivity Substrate를 사용하였다. 화학발광 신호는 LAS-3000 Luminescent image analyzer로 검출하였다.
BMMs were washed with cold phosphate buffered saline (PBS) and resuspended in 50 mM tris-hydrochloric acid (pH 8.0), 5 mM EDTA, 150 mM sodium chloride, 1% NP-40, 0.1% SDS, 1 nM PMSF, Was dissolved in a protein-extraction buffer consisting of a phosphate-buffered phosphate buffer and a phosphate-buffered phosphate buffer. Cell lysates were centrifuged at 4O < 0 > C at 10,000 xg for 15 minutes. Protein concentration was measured using a BCA protein assay kit. Protein samples (20 μg) were resuspended in sample buffer (100 nM tris-hydrochloric acid, 2% SDS, 1% 2-mercaptoethanol, 2% glycerol and 0.01% Blue, pH 7.6) and incubated for 5 minutes at 95 ° C and loaded onto a 12% polyacrylamine gel. Electrophoresis was performed using mini protean 3 cells (Bio-Rad, Hercules, Calif., USA), and proteins separated on the gel were transferred to a PVDF membrane. The membranes were incubated in blocking buffer (10 mM tris-hydrochloric acid (pH 7.5), 150 mM sodium chloride, 0.1
① HRT-BU(황련해독탕 부탄올 분획) 및 fHRT-BU(황련해독탕 발효물 부탄올 분획)의 MAPK 및 NF-kB 신호전달계에 미치는 영향(1) Effect of HRT-BU on the MAPK and NF-kB signaling systems of fHRT-BU (butanol fraction from Huangliao) and fHRT-BU
HRT(황련해독탕) 및 fHRT(황련해독탕 발효물)의 분획물이 파골세포 분화와 관련된 MAPK 및 NF-kB 신호전달계에 미치는 영향을 확인하였으며, 도 6에 결과를 나타내었다. 도 6에 나타난 바와 같이, HRT-BU와 비교하여 fHRT-BU127은 RANKL 처리로 증가되는 p38, IKKα/β 및 p65의 활성을 추가적으로 억제하였으며, fHRT-BU166은 p-ERK 및 p-JNK를 추가적으로 억제하는 것을 확인하였다.
The effect of the fraction of HRT (Huangliao Yang Taotang) and fHRT (Huangliao Taotang fermentation product) on the MAPK and NF-kB signaling system associated with osteoclast differentiation was confirmed, and the results are shown in Fig. As shown in FIG. 6, fHRT-BU127 additionally inhibited the activity of p38, IKKα / β and p65 increased by RANKL treatment, and fHRT-BU166 additionally suppressed p-ERK and p-JNK .
② HRT-BU(황련해독탕 부탄올 분획) 및 fHRT-BU(황련해독탕 발효물 부탄올 분획)의 NFATc1 및 Atp6v0d2 발현에 대한 효과(2) Effect of HRT-BU (Huangliao and Tofang Butanol Fraction) and fHRT-BU (Butanol fraction of Huangliao Yang Fungus fermented product) on NFATc1 and Atp6v0d2 expression
파골세포 분화관련 마스터 전사인자인 NFATc1과 다핵성 파골세포 형성에 관여하는 Atp6v0d2 발현에 대한 효과를 확인하였으며, 도 7에 결과를 나타내었다. 도 7에 나타난 바와 같이, NFATc1 및 Atp6v0d2의 단백질 발현이 RANKL 처리 2-3일 차에 각각 증가되는 것을 확인하였으며, HRT-BU(10 ㎍/ml)가 두 단백질의 발현 증가를 억제하며 HRT-BU와 비교하여 fHRT-BU127(10 ㎍/ml) 및 fHRT-BU166(10 ㎍/ml)이 추가적으로 두 단백질 발현을 억제하는 것을 확인하였다.
The effect of NFATc1, which is a master transcription factor related to osteoclast differentiation, and Atp6v0d2, which is involved in polycational osteoclast formation, was confirmed, and the results are shown in Fig. As shown in Fig. 7, protein expression of NFATc1 and Atp6v0d2 was increased in the 2-3 days of RANKL treatment, and HRT-BU (10 ㎍ / ml) (10 ㎍ / ml) and fHRT-BU166 (10 ㎍ / ml) inhibited the addition of two proteins.
4) real-time quantitative PCR(QPCR) 분석4) Real-time quantitative PCR (QPCR) analysis
NF-kB에 의해 조절된 유전자의 mRNA 발현에 대한 효능을 평가하기 위하여, 세포(3×105 cell/well in a 6 well plate)는 M-CSF(60 ng/ml) 존재하에 2시간 동안 시료와 배양하였다. total RNA는 RANKL(150 ng/ml)의 3, 6, 12 시간 처리 후 RNA easy mini kit로 분리하였다. first-strand cDNA는 1 ㎍ total RNA, 1 μM oligo-dT18 프라이머, RNase 10 units 및 Omniscript Reverse Transcriptase와 함께 합성하였다. 그 후, SYBR green-based QPCR 증폭은 1:3으로 희석한 first-strand cDNA 및 10 pmol 프라이머를 사용하여 SYBR Green PCR Master Mix 및 Applied Biosystems 7500 Real-time PCR system을 이용하여 수행하였다. PCR 반응은 3단계로 구성되는데, 첫 번째는 95℃에서 10분 동안 중합효소 활성화를 수행한다. 두 번째 단계는 94℃에서 30초, 60℃에서 40초 및 72℃에서 1분 동안 세 단계 사이클을 수행한다. PCR 생성물 Temperature melting curve의 생성을 위한 세 번째 단계는 95℃에서 1분, 60℃에서 30초 및 95℃에서 30초 수행된다. 모든 반응은 3번 반복하였으며, 데이터는 2-△△ CT 방법으로 분석하였다. β-엑틴(actin)은 내부표준으로 사용하였다. 분석 결과를 도 8에 나타내었다.Cells (3 × 10 5 cells / well in a 6 well plate) were incubated for 2 hours in the presence of M-CSF (60 ng / ml) to evaluate the effect of the gene regulated by NF-kB on mRNA expression ≪ / RTI > Total RNA was isolated by RNA easy mini kit after treatment with RANKL (150 ng / ml) for 3, 6 and 12 hours. First-strand cDNA was synthesized with 1 μg total RNA, 1 μM oligo-dT 18 primer,
도 8에 나타난 바와 같이, Vehicle과 비교하여 HRT-BU127이 ICAM, TNF-a 및 Nfkb2의 유전자 mRNA 발현을 추가적으로 억제하는 것을 확인하였다.
As shown in Fig. 8, it was confirmed that HRT-BU127 additionally suppressed gene mRNA expression of ICAM, TNF-a and Nfkb2 as compared with Vehicle.
5) HRT(황련해독탕) 및 fHRT(황련해독탕 발효물)의 분획의 NFATc1 과발현에 대한 효과5) Effect on the NFATc1 overexpression of the fraction of HRT (Huangliao Yangtang) and fHRT
파골세포에 전사인자인 NFATc1을 ectopic expression시키고, 이것이 HRT-BU(황련해독탕 부탄올 분획) 및 fHRT-BU(황련해독탕 발효물의 부탄올 분획)으로 인한 분화 억제효능을 극복할 수 있는지 조사하였다. HA-tagged Ca-NFATc1(the Xhol/Klenow-EcoRI 조각)을 코드화한 MSCV-CaNFATc1으로부터의 DNA 조각은 푸로마이신 선별인자(pMX-Ca-NFATc1)와 함께 pMX-puronow-EcoRI로 서브클로닝하였다. 레트로바이러스 스톡을 제조하기 위하여, pMX벡터(control) 및 pMX-Ca-NFATc1 레트로바이러스 벡터를 LiPofectamine 2000(Invitrogen)을 사용하여 PLAT-E 세포에 주입하였다. 그리고 배지는 2일 후에 수득하였으며, 이를 BMMs(1×107 cell/100 mm dish)에 M-CSF(120 ng/ml) 및 폴리브렌(polybrene, 5 ㎍/ml)와 2일 동안 추가배양하였다. 푸로마이신-저항성(puromycin-resistant)인 BMMs는 60 ng/ml M-CSF 및 150 ng/ml RANKL 존재하에 4 내지 5일 동안 파골세포로 분화하였다. 조사 결과는 도 9에 나타내었다. 도 9에 나타난 바와 같이, pMX vector를 transfection 시킨 BMM에서 Vehicle과 비교하여 HRT-BU는 78%, fHRT-BU127은 88% 그리고 fHRT-BU166은 90% TRAP 활성 억제 효능을 보였으며, HRT-BU는 93%, fHRT-BU127은 98% 그리고 fHRT-BU166은 95%의 TRAP 양성 다핵성 파골세포 형성 억제 효능을 나타내었다. 또한, pMX vector와 비교하여 Ca-NFATc1을 trasfection 시킨 BMM은 약 2배의 TRAP 활성 증가를 보였으며, Ca-NFATc1을 trasnfection 시킨 BMM과 비교하여 HRT-BU는 85%, fHRT-BU127은 90% 그리고 fHRT-BU166은 95%의 TRAP 활성 억제 효능을 보였다. 또한 pMX vector와 비교하여 Ca-NFATc1을 trasfection 시킨 BMM은 약 3배의 다핵성 파골세포 형성 증가를 보였으며, Ca-NFATc1을 transfection 시킨 BMM과 비교하여 HRT-BU는 92%, fHRT-BU127은 88% 그리고 fHRT-BU166은 98%의 TRAP 양성 다핵성 파골세포 형성 억제 효능을 나타내었다.
Ectopic expression of NFATc1, a transcription factor in osteoclasts, was examined to see if this could overcome the inhibition of differentiation by HRT-BU (Huangliao and Tofang Butanol Fraction) and fHRT-BU (Butanol Fraction of Huangliao Fungi Fermentation). DNA fragments from MSCV-CaNFATc1 encoding HA-tagged Ca-NFATc1 (the Xhol / Klenow-EcoRI fragment) were subcloned into pMX-puronow-EcoRI along with the puromycin selectin (pMX-Ca-NFATc1). To construct retroviral stock, pMX vector (control) and pMX-Ca-NFATc1 retroviral vector were injected into PLAT-E cells using LiPofectamine 2000 (Invitrogen). The medium was then harvested 2 days later and further cultured with BM-CSM (120 ng / ml) and polybrene (5 μg / ml) for 2 days in BMMs (1 × 10 7 cells / 100 mm dish) . The puromycin-resistant BMMs differentiated into osteoclasts for 4 to 5 days in the presence of 60 ng / ml M-CSF and 150 ng / ml RANKL. The results of the investigation are shown in Fig. As shown in FIG. 9, in the BMM transfected with the pMX vector, HRT-BU was inhibited by 78%, fHRT-BU127 by 88% and fHRT-BU166 by 90% 93%, fHRT-BU127 98% and fHRT-BU166 95% TRAP-positive polynuclear osteoclast formation inhibitory effect. In addition, the BMMs transfected with Ca-NFATc1 showed an increase of about 2-fold TRAP activity compared with the pMX vector. Compared with BMMs transfected with Ca-NFATc1, HRT-BU was 85%, fHRT-BU127 was 90% fHRT-BU166 showed 95% inhibition of TRAP activity. Compared with pMX vector, BM-MTM transfected with Ca-NFATc1 showed about 3-fold increase in the formation of polynuclear osteoclast. Compared with BMM transfected with Ca-NFATc1, HRT-BU was 92%, fHRT-BU127 was 88 % And fHRT-BU166 showed 98% TRAP-positive polynuclear osteoclast formation inhibitory activity.
실험예Experimental Example
2: 2:
inin
--
vivovivo
분석 analysis
1) 실험동물1) Experimental animals
190 내지 210 g의 8주된 암컷 Sprague-Dawley 랫트(Orient Bio Inc., Seoul, Korea)를 실험에 사용하였다. 실험 전 랫트는 7일간의 순화과정을 거쳐 실험에 사용하였으며, 순화과정 동안 22±1℃ 온도 및 55±10% 습도가 조절된 사육실에서 자유식이로 사육하였으며, 명암 주기는 12시간 주기로 조절하였다. 그 후 sham-operate(Sham, n=8) 또는 난소 절제술(OVX, n=56)을 실시하였다. 동물실험은 한국한의학연구원 동물실험운영규정에 준하여 취급하였다.
Male Sprague-Dawley rats (Orient Bio Inc., Seoul, Korea), weighing 190 to 210 g, were used in the experiment. The rats were housed in a control room at a temperature of 22 ± 1 ° C and humidity of 55 ± 10% during the refinement process. Then sham-operated (Sham, n = 8) or ovariectomy (OVX, n = 56) was performed. Animal experiments were handled in accordance with Korean Animal Husbandry Laboratories.
2) 시료투여 및 시료에 따른 골손실 효과 분석2) Analysis of bone loss effect according to sample administration and sample
상기 난소 절제(OVX) 랫트는 수술 일주일 후, 각 8마리씩 7군으로 나누고, 3개월 동안 황련해독탕(HRT) 및 황련해독탕 발효물(fHRT127 및 fHRT166)을 두 농도(0.3 g/kg 및 1.0 g/kg)로 경구투여 하였으며, 하기 표 2에 자세한 실험 군을 나타내었다. The ovariectomized rats (OVX) were divided into 7 groups, each of 8 rats, one week after the operation. The HRT and the fHRT126 and fHRT166 were incubated for three months at two concentrations (0.3 g / kg and 1.0 g / kg), and detailed experimental groups are shown in Table 2 below.
상기 시료투여에 따른 난소 절제(OVX) 랫트의 대퇴골에서 골손실에 대한 효과를 도 10에 나타내었다. 도 10에 나타난 바와 같이, Sham 군과 비교하여 OVX군은 골밀도(BMD)가 70% 감소, bone volume/trabecular volume(BV/TV)가 65% 감소, bone surface/bone volume raito(BS/BV)가 29% 증가, Trabecular Thinckness(Tb.Th)가 14% 감소, Trabecular separation(Tb.Sp)가 169% 증가 및 Trabecular number(Tb.N)이 60% 감소하는 효과를 나타내었다. 상기 결과를 10A 내지 도 10F를 통하여 더 자세하게 나타내었다.The effect of ovariectomized (OVX) rats on bone loss in the femur following administration of the sample is shown in Fig. As shown in FIG. 10, the OVX group showed a 70% decrease in BMD, a 65% decrease in bone volume / trabecular volume (BV / TV) , Trabecular Thinning (Tb.Th) decreased by 14%, Trabecular separation (Tb.Sp) increased by 169% and Trabecular number (Tb.N) decreased by 60%. The above results are shown in more detail through 10A to 10F.
도 10A에 나타난 바와 같이, Sham군을 100%로 하여, OVX군과 비교한 결과 fHRT127-1.0은 55% 그리고 fHRT166-0.3은 52%로 골밀도(BMD)를 유의적으로 증가시켰으며, HRT-0.3과 비교하여 fHRT166-0.3은 43%, HRT-1.0과 비교하여 fHRT127-1.0은 54%로 추가적으로 BMD를 증가키는 것을 확인하였다. As shown in FIG. 10A, when compared with the OVX group, Sham group was significantly increased by 55% in fHRT127-1.0 and 52% in fHRT166-0.3, and HRT-0.3 Compared to HRT-1.0, fHRT166-0.3 was 43%, and fHRT127-1.0 was 54%.
도 10B에 나타난 바와 같이, OVX군과 비교하여 fHRT127-1.0은 35% 그리고 fHRT166-0.3은 31%로 BV/TV를 유의적으로 증가시켰으며, HRT-0.3과 비교하여 fHRT166-0.3은 27%의 BV/TV를 증가시켰으나 유의성은 없었다. 또한, HRT-1.0과 비교하여 fHRT127-1.0은 41%로 BV/TV를 유의적이며 추가적으로 증가시켰다. As shown in FIG. 10B, compared to the OVX group, fHRT127-1.0 significantly increased BV / TV to 35% and fHRT166-0.3 to 31%, and compared to HRT-0.3, fHRT166-0.3 showed 27% BV / TV increased but not significant. In addition, compared to HRT-1.0, fHRT127-1.0 significantly increased BV / TV by 41%.
도 10C에 나타난 바와 같이, OVX군과 비교하여 fHRT127-1.0은 34%, fHRT166-0.3은 34% 그리고 fHRT166-1.0은 20%로 BS/BV를 유의적으로 감소시켰다. 또한, HRT-0.3 및 HRT-1.0과 비교하여 fHRT166-0.3 및 fHRT127-1.0은 각각 23%씩 유의적이며 추가적으로 BS/BV를 감소시켰다. As shown in FIG. 10C, BS / BV was significantly reduced by 34% in fHRT127-1.0, 34% in fHRT166-0.3, and 20% in fHRT166-1.0 compared to OVX group. In addition, compared with HRT-0.3 and HRT-1.0, fHRT166-0.3 and fHRT127-1.0 were significantly increased by 23%, respectively, further reducing BS / BV.
도 10D에 나타난 바와 같이, OVX군과 비교하여 fHRT127-1.0은 21%, fHRT166-0.3은 19% 그리고 fHRT166-1.0은 11%로 유의적으로 Tb.Th를 증가시켰으며, HRT-0.3 및 HRT-1.0과 비교하여 fHRT166-0.3 및 fHRT127-1.0은 각각 15% 유의적이며 추가적으로 Tb.Th를 증가시켰다. As shown in FIG. 10D, 21% of the fHRT127-1.0, 19% of the fHRT166-0.3, and 11% of the fHRT166-1.0 were significantly increased compared to the OVX group, and HRT-0.3 and HRT- Compared with 1.0, fHRT166-0.3 and fHRT127-1.0 were 15% significant, respectively, and additionally increased Tb.Th.
도 10E 및 도 10F에 나타난 바와 같이, OVX군과 비교하여 유의적으로 Tb.Sp 및 Tb.N을 변화시키는 군은 발견되지 않았다.
As shown in Figs. 10E and 10F, no group that significantly changed Tb.Sp and Tb.N was found compared with the OVX group.
2) Mitro-computed tomography(Micro-CT) 분석2) Mitro-computed tomography (Micro-CT) analysis
3D 골격 구조 분석을 위해, 8 ㎛ 해상도 eXplore Locus 스캐너(GEHealthcare UK Ltd., Buckinghamshire, UK)로 조직형태학적 분석을 수행하였다. 모든 형태측정 파라미터는 eXplore Micro-View(wersion 2.2, GEHealthcare)을 사용하여 계산하였다. 대퇴골에서, 스캐너 영역은 초기의 해면상의 proximal tip으로부터 약 1.7 mm 확장한 말초 골간단에 한정하였으며, 결과를 도 11에 나타내었다.
For 3D skeletal structure analysis, histomorphometric analysis was performed with an 8 μm resolution eXplore Locus Scanner (GE Healthcare UK Ltd., Buckinghamshire, UK). All morphometric parameters were calculated using eXplore Micro-View (version 2.2, GE Healthcare). At the femur, the scanner area was confined to the distal bone osteotomy extending about 1.7 mm from the initial proximal tip of the sponge, and the results are shown in Fig.
상기 모든 실험의 통계학적분석은 SPSS 소프트웨어를 사용하여 수행하였다. TRAP 활성 및 파골세포 관련 유전자의 mRNA expression level의 유의성은 β-엑틴(actin)-normalized 2-△△CT 수치를 사용한 studen's t-test로 계산하였으며, 동물실험의 통계분석을 위해, 분산의 파라메트릭 one-way 분석은 모든 다른 군 시험에 사용하였다. Duncan multiple comparison test는 그룹간 평균수치 유의차를 확인하기 위해 사용되었으며, 0.05 보다 낮은 p 수치는 의미가 있는 결과로 고려하였다.Statistical analysis of all the 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 confirm the significance of the mean difference between groups, and a p value of less than 0.05 was considered significant.
Claims (12)
A fermented product obtained by fermenting Falchelli's potash with one or more lactic acid bacteria selected from the group consisting of Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus curvatus, A pharmaceutical composition for preventing or treating osteopathy caused by reduction in bone density, comprising a fraction as an active ingredient.
[Claim 2] The pharmaceutical composition according to claim 1, wherein the medicinal herb is a hydrothermal extract of Angelica keiskei koidz.
[2] The fermented product according to claim 1, wherein the fermented product is inoculated with at least one lactic acid bacterium selected from the group consisting of Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus cabutus, Wherein the fermentation is carried out under the atmosphere at a temperature of 35 ° C to 45 ° C and for a period of 1 day to 3 days.
[Claim 2] The pharmaceutical composition according to claim 1, wherein the fraction of the fermented product of the fermented seaweed is obtained by fractionating the fermented fermented product of the fermented seaweed with ethylacetate, butanol or a mixed solvent thereof.
The pharmaceutical composition according to claim 1, wherein the bone disease caused by the decrease in bone mineral density is at least one kind of bone disease selected from the group consisting of osteoporosis, osteogenesis imperfecta, periodontal disease and fracture.
A fermented product obtained by fermenting Falchelli's potash with one or more lactic acid bacteria selected from the group consisting of Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus curvatus, A composition for health functional food for preventing or ameliorating osteopathy due to reduction in bone density, comprising a fraction as an active ingredient.
8. The health functional food composition according to claim 7, wherein the phytophthora is a hydrothermal extract of phloem, golden, yellow, white and ginger.
[7] The fermented product according to claim 7, wherein the fermented product of the fermented soybean flour is inoculated with at least one lactic acid bacterium selected from the group consisting of Lactobacillus casei, Lactobacillus flutarium, and Lactobacillus cabutus, Wherein the composition is fermented under an atmosphere at a temperature of 35 ° C to 45 ° C and for a period of 1 day to 3 days.
The health functional food composition according to claim 7, wherein the fraction of the fermented product of the fermented seaweed is obtained by fractionating the fermented product fermented by the fermented seaweed with ethylacetate, butanol or a mixed solvent thereof.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020120056355A KR101402926B1 (en) | 2012-05-25 | 2012-05-25 | Pharmaceutical composition and functional food for prevention or treatment of bone disease comprising the lactic acid bacteria ferment of hwangryunhaedoktang |
PCT/KR2012/004290 WO2013176327A1 (en) | 2012-05-25 | 2012-05-31 | 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 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020120056355A KR101402926B1 (en) | 2012-05-25 | 2012-05-25 | Pharmaceutical composition and functional food for prevention or treatment of bone disease comprising the lactic acid bacteria ferment of hwangryunhaedoktang |
Publications (2)
Publication Number | Publication Date |
---|---|
KR20130132172A KR20130132172A (en) | 2013-12-04 |
KR101402926B1 true KR101402926B1 (en) | 2014-06-03 |
Family
ID=49623994
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
KR1020120056355A KR101402926B1 (en) | 2012-05-25 | 2012-05-25 | Pharmaceutical composition and functional food for prevention or treatment of bone disease comprising the lactic acid bacteria ferment of hwangryunhaedoktang |
Country Status (2)
Country | Link |
---|---|
KR (1) | KR101402926B1 (en) |
WO (1) | WO2013176327A1 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103735719A (en) * | 2014-01-12 | 2014-04-23 | 万世凤 | Method for preparing compound coptis root capsule |
WO2019050063A1 (en) * | 2017-09-07 | 2019-03-14 | 태이생명과학 주식회사 | Composition for preventing or treating periodontal diseases comprising as active ingredient scutellaria baicalensis extract treated with enzyme isolated from aspergillus niger |
KR102144838B1 (en) * | 2019-04-18 | 2020-08-14 | 전남대학교산학협력단 | Composition for improving, preventing or treating bone diseases comprising Lactobacillus curvatus WiKim38 or culture medium thereof |
KR102199934B1 (en) * | 2019-06-26 | 2021-01-08 | 한국식품연구원 | Cosmetic compositions containing Hwangryunhaedoktang fermented with Lactobacillus plantarum WiKim0111 for anti-acne |
KR102350437B1 (en) * | 2019-06-26 | 2022-01-14 | 한국식품연구원 | Cosmetic compositions containing Hwangryunhaedoktang fermented with Lactobacillus plantarum WiKim0111 for anti-acne |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100740565B1 (en) * | 2006-01-25 | 2007-07-18 | 계명대학교 산학협력단 | Inhibition effects of scutellaria baicalensis geogri. on osteoclast differentiation |
KR100700480B1 (en) * | 2006-02-24 | 2007-03-28 | 경희대학교 산학협력단 | Composition comprising the extract of Phellodendron amurensis for preventing and treating fracture |
KR101123153B1 (en) * | 2009-10-16 | 2012-03-19 | 안국약품 주식회사 | Composition for preventing or treating periodontal diseases containing herbal extract |
KR101199961B1 (en) * | 2010-06-23 | 2012-11-12 | 한국 한의학 연구원 | Composition for Prevention or Treatment of Osteoporosis Comprising Bangpungtongseungsan and Fermentation Product Thereof with Lactic Acid Bacteria |
-
2012
- 2012-05-25 KR KR1020120056355A patent/KR101402926B1/en active IP Right Grant
- 2012-05-31 WO PCT/KR2012/004290 patent/WO2013176327A1/en active Application Filing
Non-Patent Citations (8)
Title |
---|
Arch Pharm Res, Vol.26, No.11, pp.929-936 (2003) * |
Arch Pharm Res, Vol.26, No.11, pp.929-936 (2003)* |
Biol. Pharm. Bull., Vol.33, pp.10, pp.1733-1739 (2010) * |
Biol. Pharm. Bull., Vol.33, pp.10, pp.1733-1739 (2010)* |
JOURNAL OF BONE AND MINERAL RESEARCH, Vol.23, No.8, pp.1227-1237 (2008) * |
JOURNAL OF BONE AND MINERAL RESEARCH, Vol.23, No.8, pp.1227-1237 (2008)* |
Yakhak Hoeji, Vol.55, No.1, pp.64-68 (2011) * |
Yakhak Hoeji, Vol.55, No.1, pp.64-68 (2011)* |
Also Published As
Publication number | Publication date |
---|---|
WO2013176327A1 (en) | 2013-11-28 |
KR20130132172A (en) | 2013-12-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR101494279B1 (en) | Lactobacillus plantarum KY1032 having inhibitory activity against adipocyte-specific gene expression and adipocyte differentiation, and product containing thereof as an effective factor | |
KR102206628B1 (en) | Novel Lactobacillus rhamnosus strain for preventing or treating obesity and the use thereof | |
KR20180103772A (en) | Composition for preventing or treating immune diseases comprising mixture of lactic acid bacteria | |
CN110393728B (en) | Lactobacillus, pharmaceutical composition thereof and use of edible composition in treating, preventing or improving bone diseases | |
KR101402926B1 (en) | Pharmaceutical composition and functional food for prevention or treatment of bone disease comprising the lactic acid bacteria ferment of hwangryunhaedoktang | |
KR101252639B1 (en) | Composition for Prevention or Treatment of Osteoporosis Comprising Ssanghwatang and Fermentation Product Thereof with Lactic Acid Bacteria | |
KR101800632B1 (en) | Pharmaceutical composition, food composition or food additives for prevention, improvement or treatment of muscle loss, weakening, and atrophy comprising Enterococcus faecalis, it culture broth or heat killed Enterococcus faecalis as an active ingredient | |
KR20140000620A (en) | Probiotic strains for use in improving the enteric nervous system | |
EP4364745A2 (en) | Novel lactic acid bacteria and use thereof | |
KR102242668B1 (en) | Composition for improving, preventing or treating bone diseases comprising Lactobacillus sakei CVL-001 or culture medium thereof | |
KR20120112510A (en) | Dna damage repair promoter for oral application, and elastase activity inhibitor for oral application | |
KR102420930B1 (en) | Composition for preventing and treating of obesity comprising powder of lactic acid cell lysate | |
EP4364583A1 (en) | Composition comprising three lactobacillus sp. strains, and use thereof | |
KR101770035B1 (en) | Composition comprising Morifolium extract asan effective component for preventing and treatingarthritis | |
KR101709281B1 (en) | Composition for Prevention or Treatment of Osteoporosis Comprising Herbal Extract and Fermentation Product thereof with Lactic acid Bacteria | |
KR101770036B1 (en) | Composition comprising Schizandrae Fructus extract asan effective component for preventing and treatingarthritis | |
KR20130138037A (en) | Lactic acid bacteria ferment of bojungikkitang and use thereof | |
EP3932416A2 (en) | Composition for improving, preventing, or treating bone diseases or metabolic diseases, including novel lactobacillus sakei cvl-001 strain and culture medium thereof | |
KR101577732B1 (en) | Pharmaceutical composition comprising ferment of Phellodendron amurensis as an active ingredient for prevention or treatment of metabolic bone disease or climacteric disease | |
KR101457117B1 (en) | Pharmaceutical composition and functional food for prevention or treatment of bone disease comprising the dryopteris crassithizoma extract | |
KR102144838B1 (en) | Composition for improving, preventing or treating bone diseases comprising Lactobacillus curvatus WiKim38 or culture medium thereof | |
KR20140100608A (en) | Composition for preventing and improving andropause syndrome and aging of male comprising rooibos extract as an active ingredient | |
KR101554875B1 (en) | Lactobacillus plantarum yd-212 strains, and composition containing fermentation substance prepared by using the same | |
KR20200047939A (en) | Anti-diabetic and Anti-obese Fermented Composition Comprising Fermented Hot Pepper and Fermented Hot Pepper Paste | |
KR101535077B1 (en) | The products containing probiotic Bifidobacterium lactis HY8101 having activity preventing from insulin resistance, which caused type 2 diabetes mellitus |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A201 | Request for examination | ||
E902 | Notification of reason for refusal | ||
E701 | Decision to grant or registration of patent right | ||
GRNT | Written decision to grant | ||
FPAY | Annual fee payment |
Payment date: 20170412 Year of fee payment: 4 |
|
FPAY | Annual fee payment |
Payment date: 20180410 Year of fee payment: 5 |