KR20210006801A - Composition for improving bone metabolism comprising shitake mushroom extracts - Google Patents
Composition for improving bone metabolism comprising shitake mushroom extracts Download PDFInfo
- Publication number
- KR20210006801A KR20210006801A KR1020190082873A KR20190082873A KR20210006801A KR 20210006801 A KR20210006801 A KR 20210006801A KR 1020190082873 A KR1020190082873 A KR 1020190082873A KR 20190082873 A KR20190082873 A KR 20190082873A KR 20210006801 A KR20210006801 A KR 20210006801A
- Authority
- KR
- South Korea
- Prior art keywords
- bone
- extract
- composition
- shiitake
- treated
- Prior art date
Links
- 239000000284 extract Substances 0.000 title claims abstract description 139
- 239000000203 mixture Substances 0.000 title claims abstract description 40
- 230000004097 bone metabolism Effects 0.000 title claims abstract description 26
- 235000001674 Agaricus brunnescens Nutrition 0.000 title description 16
- 240000000599 Lentinula edodes Species 0.000 claims abstract description 110
- 235000013305 food Nutrition 0.000 claims abstract description 8
- 201000010099 disease Diseases 0.000 claims abstract description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 7
- 210000000988 bone and bone Anatomy 0.000 claims description 83
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 82
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims description 11
- 238000000605 extraction Methods 0.000 claims description 10
- 239000004480 active ingredient Substances 0.000 claims description 7
- 150000005846 sugar alcohols Polymers 0.000 claims description 7
- 230000006378 damage Effects 0.000 claims description 6
- 125000004432 carbon atom Chemical group C* 0.000 claims description 4
- 208000037824 growth disorder Diseases 0.000 claims description 4
- 206010068975 Bone atrophy Diseases 0.000 claims description 3
- 208000037147 Hypercalcaemia Diseases 0.000 claims description 3
- 208000029725 Metabolic bone disease Diseases 0.000 claims description 3
- 208000010191 Osteitis Deformans Diseases 0.000 claims description 3
- 206010049088 Osteopenia Diseases 0.000 claims description 3
- 208000001132 Osteoporosis Diseases 0.000 claims description 3
- 208000027868 Paget disease Diseases 0.000 claims description 3
- 201000010103 fibrous dysplasia Diseases 0.000 claims description 3
- 230000000148 hypercalcaemia Effects 0.000 claims description 3
- 208000030915 hypercalcemia disease Diseases 0.000 claims description 3
- 208000027202 mammary Paget disease Diseases 0.000 claims description 3
- 230000001613 neoplastic effect Effects 0.000 claims description 3
- 208000005368 osteomalacia Diseases 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 82
- 239000003814 drug Substances 0.000 abstract description 6
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 229930014626 natural product Natural products 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 208000018522 Gastrointestinal disease Diseases 0.000 abstract description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 53
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 53
- 210000004027 cell Anatomy 0.000 description 38
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 33
- 229960005069 calcium Drugs 0.000 description 33
- 239000011575 calcium Substances 0.000 description 33
- 229910052791 calcium Inorganic materials 0.000 description 33
- 102000004169 proteins and genes Human genes 0.000 description 32
- 108090000623 proteins and genes Proteins 0.000 description 32
- 239000000758 substrate Substances 0.000 description 31
- 238000002835 absorbance Methods 0.000 description 30
- 235000018102 proteins Nutrition 0.000 description 27
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 26
- 210000000963 osteoblast Anatomy 0.000 description 24
- 229940088594 vitamin Drugs 0.000 description 24
- 229930003231 vitamin Natural products 0.000 description 24
- 235000013343 vitamin Nutrition 0.000 description 24
- 239000011782 vitamin Substances 0.000 description 24
- 150000003722 vitamin derivatives Chemical class 0.000 description 24
- 238000012360 testing method Methods 0.000 description 19
- 102000005600 Cathepsins Human genes 0.000 description 17
- 108010084457 Cathepsins Proteins 0.000 description 17
- 102000004067 Osteocalcin Human genes 0.000 description 16
- 108090000573 Osteocalcin Proteins 0.000 description 16
- 230000015572 biosynthetic process Effects 0.000 description 16
- 102000008186 Collagen Human genes 0.000 description 15
- 108010035532 Collagen Proteins 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 15
- 229920001436 collagen Polymers 0.000 description 15
- 230000011164 ossification Effects 0.000 description 15
- 210000001519 tissue Anatomy 0.000 description 14
- 238000010835 comparative analysis Methods 0.000 description 12
- 238000003786 synthesis reaction Methods 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 235000001715 Lentinula edodes Nutrition 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 229940088598 enzyme Drugs 0.000 description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 9
- 239000013642 negative control Substances 0.000 description 9
- 239000013641 positive control Substances 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 230000003833 cell viability Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 230000018678 bone mineralization Effects 0.000 description 7
- 230000004663 cell proliferation Effects 0.000 description 7
- 230000004069 differentiation Effects 0.000 description 7
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 6
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 6
- 235000013376 functional food Nutrition 0.000 description 6
- 230000028327 secretion Effects 0.000 description 6
- 239000003826 tablet Substances 0.000 description 6
- 102000004171 Cathepsin K Human genes 0.000 description 5
- 108090000625 Cathepsin K Proteins 0.000 description 5
- 230000002308 calcification Effects 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 208000006386 Bone Resorption Diseases 0.000 description 4
- 208000020084 Bone disease Diseases 0.000 description 4
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 description 4
- 102000012422 Collagen Type I Human genes 0.000 description 4
- 108010022452 Collagen Type I Proteins 0.000 description 4
- 229930003316 Vitamin D Natural products 0.000 description 4
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products 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-XFEUOLMDSA-N 0.000 description 4
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 4
- 235000013361 beverage Nutrition 0.000 description 4
- 230000008468 bone growth Effects 0.000 description 4
- 230000024279 bone resorption Effects 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 230000036541 health Effects 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
- 238000005259 measurement Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 235000016709 nutrition Nutrition 0.000 description 4
- 230000035764 nutrition Effects 0.000 description 4
- 230000004072 osteoblast differentiation Effects 0.000 description 4
- 210000002997 osteoclast Anatomy 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 4
- 235000019166 vitamin D Nutrition 0.000 description 4
- 239000011710 vitamin D Substances 0.000 description 4
- 150000003710 vitamin D derivatives Chemical class 0.000 description 4
- 229940046008 vitamin d Drugs 0.000 description 4
- 102000015775 Core Binding Factor Alpha 1 Subunit Human genes 0.000 description 3
- 108010024682 Core Binding Factor Alpha 1 Subunit Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000002033 PVDF binder Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 235000015165 citric acid Nutrition 0.000 description 3
- 235000005911 diet Nutrition 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 238000012933 kinetic analysis Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000012139 lysis buffer Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 2
- 108010049931 Bone Morphogenetic Protein 2 Proteins 0.000 description 2
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 description 2
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- DHCLVCXQIBBOPH-UHFFFAOYSA-N Glycerol 2-phosphate Chemical compound OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 2
- 241000202807 Glycyrrhiza Species 0.000 description 2
- 235000001453 Glycyrrhiza echinata Nutrition 0.000 description 2
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 description 2
- 235000017382 Glycyrrhiza lepidota Nutrition 0.000 description 2
- 101000762366 Homo sapiens Bone morphogenetic protein 2 Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 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
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- DLRVVLDZNNYCBX-UHFFFAOYSA-N Polydextrose Polymers OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(O)O1 DLRVVLDZNNYCBX-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
- MECHNRXZTMCUDQ-UHFFFAOYSA-N Vitamin D2 Natural products C1CCC2(C)C(C(C)C=CC(C)C(C)C)CCC2C1=CC=C1CC(O)CCC1=C MECHNRXZTMCUDQ-UHFFFAOYSA-N 0.000 description 2
- 230000009102 absorption Effects 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 230000033558 biomineral tissue development Effects 0.000 description 2
- 210000002449 bone cell Anatomy 0.000 description 2
- 230000037182 bone density Effects 0.000 description 2
- 229940112869 bone morphogenetic protein Drugs 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 235000008504 concentrate Nutrition 0.000 description 2
- 210000002808 connective tissue Anatomy 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- VIYFPAMJCJLZKD-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate Chemical compound [Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 VIYFPAMJCJLZKD-UHFFFAOYSA-L 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 229960002061 ergocalciferol Drugs 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000003599 food sweetener Nutrition 0.000 description 2
- -1 for example Substances 0.000 description 2
- 239000003205 fragrance Substances 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 229940010454 licorice Drugs 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical class OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 239000010421 standard material Substances 0.000 description 2
- 239000003765 sweetening agent Substances 0.000 description 2
- 229960003080 taurine Drugs 0.000 description 2
- 235000019154 vitamin C Nutrition 0.000 description 2
- 239000011718 vitamin C Substances 0.000 description 2
- 235000001892 vitamin D2 Nutrition 0.000 description 2
- 239000011653 vitamin D2 Substances 0.000 description 2
- MECHNRXZTMCUDQ-RKHKHRCZSA-N vitamin D2 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)/C=C/[C@H](C)C(C)C)=C\C=C1\C[C@@H](O)CCC1=C MECHNRXZTMCUDQ-RKHKHRCZSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000000080 wetting agent Substances 0.000 description 2
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 description 1
- NOOLISFMXDJSKH-KXUCPTDWSA-N (-)-Menthol Chemical compound CC(C)[C@@H]1CC[C@@H](C)C[C@H]1O NOOLISFMXDJSKH-KXUCPTDWSA-N 0.000 description 1
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- 229940043375 1,5-pentanediol Drugs 0.000 description 1
- QAPSNMNOIOSXSQ-YNEHKIRRSA-N 1-[(2r,4s,5r)-4-[tert-butyl(dimethyl)silyl]oxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O[Si](C)(C)C(C)(C)C)C1 QAPSNMNOIOSXSQ-YNEHKIRRSA-N 0.000 description 1
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108010011485 Aspartame Proteins 0.000 description 1
- 208000018083 Bone metabolism disease Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines 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-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
- 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
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000013382 Gelatinases Human genes 0.000 description 1
- 108010026132 Gelatinases Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 229920002907 Guar gum Polymers 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 208000012659 Joint disease Diseases 0.000 description 1
- 206010023509 Kyphosis Diseases 0.000 description 1
- MIJPAVRNWPDMOR-ZAFYKAAXSA-N L-ascorbic acid 2-phosphate Chemical compound OC[C@H](O)[C@H]1OC(=O)C(OP(O)(O)=O)=C1O MIJPAVRNWPDMOR-ZAFYKAAXSA-N 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 241000222350 Pleurotus Species 0.000 description 1
- 229920001100 Polydextrose Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 235000014443 Pyrus communis Nutrition 0.000 description 1
- 241000269821 Scombridae Species 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- 102000011117 Transforming Growth Factor beta2 Human genes 0.000 description 1
- 101800000304 Transforming growth factor beta-2 Proteins 0.000 description 1
- 229930003451 Vitamin B1 Natural products 0.000 description 1
- 108091005605 Vitamin K-dependent proteins 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
- 229960000583 acetic acid Drugs 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000010478 bone regeneration Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 230000004094 calcium homeostasis Effects 0.000 description 1
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 description 1
- 239000001527 calcium lactate Substances 0.000 description 1
- 235000011086 calcium lactate Nutrition 0.000 description 1
- 229960002401 calcium lactate Drugs 0.000 description 1
- 230000003913 calcium metabolism Effects 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
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229960001927 cetylpyridinium chloride Drugs 0.000 description 1
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 229960004106 citric acid Drugs 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 229940009976 deoxycholate Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 239000002934 diuretic Substances 0.000 description 1
- 230000001882 diuretic effect Effects 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 230000000480 effect on calcification Effects 0.000 description 1
- 238000001378 electrochemiluminescence detection Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 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
- 238000011156 evaluation Methods 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000020510 functional beverage Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000000665 guar gum Substances 0.000 description 1
- 235000010417 guar gum Nutrition 0.000 description 1
- 229960002154 guar gum Drugs 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 208000024798 heartburn Diseases 0.000 description 1
- 235000012907 honey Nutrition 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000012625 in-situ measurement Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 235000020640 mackerel Nutrition 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 239000008204 material by function Substances 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
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 201000008383 nephritis Diseases 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- VSMOENVRRABVKN-UHFFFAOYSA-N oct-1-en-3-ol Chemical compound CCCCCC(O)C=C VSMOENVRRABVKN-UHFFFAOYSA-N 0.000 description 1
- 235000021032 oily fish Nutrition 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000003071 parasitic effect Effects 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
- 239000001814 pectin Substances 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 229960000292 pectin Drugs 0.000 description 1
- WCVRQHFDJLLWFE-UHFFFAOYSA-N pentane-1,2-diol Chemical compound CCCC(O)CO WCVRQHFDJLLWFE-UHFFFAOYSA-N 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000003616 phosphatase activity assay Methods 0.000 description 1
- 230000037081 physical activity Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000001259 polydextrose Substances 0.000 description 1
- 235000013856 polydextrose Nutrition 0.000 description 1
- 229940035035 polydextrose Drugs 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000009103 reabsorption Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 229940109850 royal jelly Drugs 0.000 description 1
- 235000019204 saccharin Nutrition 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 229940081974 saccharin Drugs 0.000 description 1
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229940044203 shiitake mushroom preparation Drugs 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 238000013424 sirius red staining Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- 229960003885 sodium benzoate Drugs 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000011775 sodium fluoride Substances 0.000 description 1
- 235000013024 sodium fluoride Nutrition 0.000 description 1
- CMZUMMUJMWNLFH-UHFFFAOYSA-N sodium metavanadate Chemical compound [Na+].[O-][V](=O)=O CMZUMMUJMWNLFH-UHFFFAOYSA-N 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 229960002920 sorbitol Drugs 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 229940072041 transforming growth factor beta 2 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
- 210000000689 upper leg Anatomy 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 230000036448 vitalisation Effects 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 238000001262 western blot Methods 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
- 229910000166 zirconium phosphate Inorganic materials 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
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
-
- 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
-
- 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
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
-
- 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
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
- A23V2250/208—Fungi extracts
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Mycology (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Physical Education & Sports Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
Description
본 발명은 표고버섯의 용매 추출물을 포함하는 골 대사 개선용 조성물에 관한 것이다.The present invention relates to a composition for improving bone metabolism comprising a solvent extract of shiitake mushrooms.
뼈는 특별한 연결 조직이며 골격시스템을 형성하여 신체를 지지하고 근육활동을 용이하게 하며 내장기관을 보호한다. 뼈의 구성은 인산칼슘복염과 여러 이온들의 석회화로 이루어지고 콜라겐, 당단백들이 주요 구성물로 포함되어 있다.Bone is a special connective tissue and forms a skeletal system to support the body, facilitate muscle activity, and protect internal organs. The composition of bone consists of calcium phosphate double salt and calcification of various ions, and collagen and glycoproteins are included as major components.
조골세포가 뼈 표면에서 골형성을 조절하고 파골세포가 뼈 표면의 복구와 골흡수를 조절하여 골형성/골흡수 시스템의 작용을 통해 뼈의 성장 및 재생을 담당하며, cytokine, 칼슘, 인, 호르몬 등과 같은 다양한 인자들에 의해 조절된다.Osteoblasts control bone formation on the bone surface, and osteoclasts control bone surface repair and bone resorption, and are responsible for bone growth and regeneration through the action of the bone formation/bone resorption system, and cytokine, calcium, phosphorus, hormones It is controlled by a variety of factors, such as.
성장기의 뼈 성장은 골형성과 골흡수의 적절한 균형에 의해 이루어지며 사춘기와 성인기 초반에는 신체적 활동과 식이 요인이 중요하고, 특히 칼슘 섭취량이 최대 골밀도를 증가시킨다고 보고된바 있다.Bone growth during the growth phase is achieved by the proper balance of bone formation and bone resorption, and physical activity and dietary factors are important in puberty and early adulthood, and it has been reported that calcium intake increases the maximum bone density.
40대 이후 분해되어 체내로 흡수되는 양이 형성되는 양보다 점차 증가하면서 골량이 감소하게 되며, 특히 노인은 골밀도의 급격한 감소로 굽은 허리, 척추후만증, 골절, 골관절 질환 등의 유발 위험성이 크다.Bone mass decreases as the amount that is decomposed and absorbed into the body increases gradually after the 40s, and in particular, the elderly have a high risk of causing curved waist, kyphosis, fracture, and bone joint disease due to a sharp decrease in bone density.
2026년 한국의 65세 이상 인구비율이 20%, 2050년에 그 비율이 38%로 올라 초고령화사회에 진입하면서 의료비 증가에 대한 개인과 국가의 의료비용 부담이 가중될 것으로 예측되어 골관련 질환을 예방하고 개선을 기대할 수 있는 건강기능식품에 대한 선호도는 점차 확대될 것으로 전망된다.Bone-related diseases are expected to increase as the proportion of the population aged 65 and over in Korea rises to 20% in 2026 and 38% in 2050, entering an ultra-aging society, increasing the burden of medical expenses on individuals and countries. The preference for health functional foods that can be prevented and improved is expected to gradually expand.
비타민 D는 호르몬 전구체로서 Ergocalciferol, 즉 vitamin D2는 버섯 등의 일부 식물과 고등어와 같은 기름진 생선 등에 존재하며, 식이 섭취를 통해 섭취되는 비타민 D는 체내에서 칼슘의 항상성과 뼈의 대사를 조절하는데 혈청 칼슘이 감소하면 부갑상샘 호르몬이 분비되고 이것은 비타민D를 활성형 비타민D로 전환시켜 장에서의 칼슘 흡수 증가, 신장에서의 칼슘 재흡수 촉진, 뼈 흡수 등의 작용을 한다고 알려져 있다.Vitamin D is a hormone precursor, and Ergocalciferol, or vitamin D2, is present in some plants such as mushrooms and oily fish such as mackerel, and vitamin D ingested through diet regulates calcium homeostasis and bone metabolism in the body. When this decreases, parathyroid hormone is secreted, which converts vitamin D into active vitamin D, which is known to increase calcium absorption in the intestine, promote calcium reabsorption in the kidneys, and absorb bones.
조골세포와 파골세포의 신호조절을 통하여 골질환을 예방하고 치료할 수 있는 천연물 신약은 현재까지 거의 없으므로 골대사 조절을 통해 골질환을 예방 및 개선할 수 있는 천연물 소재발굴 기술이 중요한 시점이다.Since there are few new natural drugs that can prevent and treat bone diseases through the signal control of osteoblasts and osteoclasts so far, it is an important time to develop technologies for natural products that can prevent and improve bone diseases through control of bone metabolism.
따라서, 천연물 소재를 탐색하여 골대사 개선을 위한 기능성 물질의 개발이 지속적으로 요구되고 있다.Therefore, there is a continuous demand for the development of functional materials for improving bone metabolism by searching for natural materials.
본 발명자들은 천연물로부터 유익한 성분들을 최적으로 추출하여 이를 유익한 기능성 식품으로 개발하고자 예의 연구한 결과, 후술하는 바와 같이 표고버섯 추출물이 골 대사를 개선시킬 수 있음을 예기치 않게 발견하고 이에 관련 실험을 통해 효능을 입증함으로써 본 발명을 완성하기에 이르렀다.As a result of intensive research to develop beneficial functional foods by optimally extracting beneficial ingredients from natural products, the present inventors unexpectedly discovered that shiitake mushroom extract can improve bone metabolism as described later, and through related experiments, efficacy By demonstrating, the present invention was completed.
따라서, 본 발명의 목적은, 일면에 있어서, 표고버섯 추출물을 유효 성분으로 포함하는 것을 특징으로 하는 골 대사 개선용 조성물을 제공하는데에 있다.Accordingly, an object of the present invention is to provide, in one aspect, a composition for improving bone metabolism, comprising a shiitake mushroom extract as an active ingredient.
본 발명에 따른 표고버섯 추출물을 포함하는 골 대사 개선용 조성물은 골 대사에 기인하는 질환에 유익한 효능을 나타내는 것이 확인되고, 천연물 유래의 추출물로서 기존의 약제를 장기복용 했을 때 문제점으로 지적되는 위장장애 및 속쓰림 등의 부작용을 최소화할 수 있다는 것도 장점으로 부각됨에 따라 이를 요하는 식품 또는 의약으로 유용하게 이용될 수 있다.The composition for improving bone metabolism comprising the shiitake mushroom extract according to the present invention has been confirmed to exhibit beneficial effects on diseases caused by bone metabolism, and gastrointestinal disorders pointed out as a problem when long-term use of existing drugs as extracts derived from natural products And it is also possible to minimize side effects such as heartburn, as it emerges as an advantage, it can be usefully used as a food or medicine that requires it.
도 1은 특이 기질에 대한 알칼리 포스파타아제의 표준 그래프이다.
도 2는 특이 기질에 대한 각 시료들의 평균 흡광도를 나타낸 그라프도이다.
도 3은 각 시료의 평균 알칼리 포스파타아제 단위이다.
도 4는 특이 기질에 대한 시료의 평균 Vmax 값을 나타내는 그라프도이다.
도 5는 표고버섯 열수 추출물의 평균 칼슘 레벨을 나타내는 그라프도이다.
도 6은 시료의 특이 기질에 대한 평균 흡광도를 나타내는 그라프도이다.
도 7은 시료의 특이 기질에 대한 평균 카텝신 환산 단위를 나타내는 그라프도이다
도 8은 시료의 특이 기질에 대한 평균 Vmax 값을 나타내는 그라프도이다.
도 9는 시료의 평균 Vmax 값의 백분율을 나타내는 그라프도이다.
도 10은 조직 균질화물에 대한 열수 추출물의 NO 레벨을 나타내는 그라프도이다.
도 11은 조직 균질화물에 대한 열수 추출물의 NO 레벨의 백분율을 나타내는 그라프도이다.
도 12는 특이 기질에 대한 시료의 평균 흡광도를 나타내는 그라프도이다.
도 13은 특이 기질에 대한 시료의 평균 알칼리 포스파타아제 단위를 나타내는 그라프도이다.
도 14는 조직 균질화물에 대한 시료의 평균 칼슘 레벨을 나타내는 그라프도이다.
도 15는 조직 균질화물에 대한 열수 추출물의 평균 단백질 농도를 나타내는 그라프도이다.
도 16은 조직 균질화물에 대한 열수 추출물의 평균 단백질 농도의 백분율을 나타내는 그라프도이다.
도 17은 MC 3T3-E1 세포주에 대한 표고버섯 추출물의 세포 생존능에 대한 영향을 나타내는 그라프도이다.
도 18은 MC 3T3-E1 세포주에 대한 비타민D-표고버섯 추출물의 세포 생존능에 대한 영향을 나타내는 그라프도이다.
도 19는 MC 3T3-E1 세포주에 대한 표고버섯 추출물의 세포 증식에 대한 영향을 나타내는 그라프도이다.
도 20은 MC 3T3-E1 세포주에 대한 비타민D-표고버섯 추출물의 세포 증식에 대한 영향을 나타내는 그라프도이다.
도 21은 표고버섯 추출물의 MC3T3-E1 세포주에서 골석회화 형성능에 대한 영양을 나타내는 그라프도이다.
도 22는 비타민D-표고버섯 추출물의 MC3T3-E1 세포주에서 골석회화 형성능에 대한 영양을 나타내는 그라프도이다.
도 23은 MC3T3-E1 세포주에서 ALP 활성에 관한 표고버섯 추출물의 영향을 나타내는 그라프도이다.
도 24는 MC3T3-E1 세포주에서 ALP 활성에 관한 비타민D-표고버섯 추출물의 영향을 나타내는 그라프도이다.
도 25는 MC3T3-E1 세포주에서 콜라겐 합성에 미치는 추출물의 영향을 나타낸 그라프도이다.
도 26은 MC3T3-E1 세포주에서 콜라겐 합성에 미치는 비타민D-추출물의 영향을 나타낸 그라프도이다.
도 27은 표고버섯 추출물의 MC3T3-E1 세포주에서 오스테오칼신 분비에 관한 영향을 나타내는 그라프도이다.
도 28은 비타민D-표고버섯 추출물의 MC3T3-E1 세포주에서 오스테오칼신 분비에 관한 영향을 나타내는 그라프도이다.
도 29는 MC3T3-E1 세포주에서 오스테오칼신 분비에 관한 비타민D-추출물의 영향을 나타낸 사진이다.1 is a standard graph of alkaline phosphatase for specific substrates.
2 is a graph showing the average absorbance of each sample for a specific substrate.
3 is an average alkaline phosphatase unit of each sample.
4 is a graph showing the average Vmax value of a sample for a specific substrate.
5 is a graph showing the average calcium level of hot water extract of shiitake mushrooms.
6 is a graph showing the average absorbance for a specific substrate of a sample.
7 is a graph showing the average cathepsin conversion unit for a specific substrate of a sample.
8 is a graph showing the average Vmax value for a specific substrate of a sample.
9 is a graph showing the percentage of an average Vmax value of a sample.
10 is a graph showing the NO level of hot water extract for tissue homogenates.
11 is a graph showing the percentage of NO levels of hot water extract with respect to tissue homogenates.
12 is a graph showing the average absorbance of a sample for a specific substrate.
13 is a graph showing the average alkaline phosphatase units of a sample against a specific substrate.
14 is a graph showing the average calcium level of a sample with respect to a tissue homogenate.
15 is a graph showing the average protein concentration of hot water extracts for tissue homogenates.
16 is a graph showing the percentage of the average protein concentration of the hot water extract with respect to the tissue homogenate.
17 is a graph showing the effect of shiitake mushroom extract on cell viability against MC 3T3-E1 cell line.
18 is a graph showing the effect of vitamin D-shiitake mushroom extract on cell viability against MC 3T3-E1 cell line.
19 is a graph showing the effect of shiitake mushroom extract on cell proliferation against MC 3T3-E1 cell line.
20 is a graph showing the effect of vitamin D-shiitake mushroom extract on cell proliferation against MC 3T3-E1 cell line.
Fig. 21 is a graph showing the nutrition of shiitake mushroom extract on the ability to form osteocalcification in the MC3T3-E1 cell line.
Figure 22 is a graph showing the nutrition of the vitamin D-shiitake mushroom extract on the ability to form bone calcium in the MC3T3-E1 cell line.
23 is a graph showing the effect of shiitake mushroom extract on ALP activity in MC3T3-E1 cell line.
24 is a graph showing the effect of vitamin D-shiitake mushroom extract on ALP activity in MC3T3-E1 cell line.
25 is a graph showing the effect of the extract on collagen synthesis in the MC3T3-E1 cell line.
26 is a graph showing the effect of vitamin D-extract on collagen synthesis in MC3T3-E1 cell line.
27 is a graph showing the effect of shiitake mushroom extract on the secretion of osteocalcin in the MC3T3-E1 cell line.
28 is a graph showing the effect of vitamin D-shiitake mushroom extract on the secretion of osteocalcin in the MC3T3-E1 cell line.
29 is a photograph showing the effect of vitamin D-extract on the secretion of osteocalcin in MC3T3-E1 cell line.
본 발명은, 일면에 있어서, 표고버섯의 용매 추출물을 유효 성분으로 포함하는 것을 특징으로 하는 골 대사 개선용 조성물을 제공한다.In one aspect, the present invention provides a composition for improving bone metabolism, comprising a solvent extract of shiitake mushrooms as an active ingredient.
본 발명은, 추가의 일면에 있어서,The present invention, in a further aspect,
상기 표고버섯은 깨끗이 세척하여 건조하여 세절한 다음 식물 원료 중량의 5배 내지 20배의 추출 용매를 더하여 고압멸균솥에 넣고 65 ~ 125℃의 온도에서 2 내지 12시간 동안 가열시켜 추출하여 여과하고 농축하여 얻은 것을 특징으로 하는 골 대사 개선용 조성물을 제공한다.The shiitake mushrooms are washed, dried, and minced, and then added to an extraction solvent of 5 to 20 times the weight of the plant material, put in an autoclave, heated at a temperature of 65 to 125°C for 2 to 12 hours, extracted, filtered, and concentrated. It provides a composition for improving bone metabolism, characterized in that obtained by.
본 발명은, 추가의 일면에 있어서,The present invention, in a further aspect,
상기 추출용매는 물, 탄소수 1 내지 4의 저급 알코올, 다가 알코올 또는 이들의 혼합물로부터 선택된 적어도 어느 하나인 것을 특징으로 하는 골 대사 개선용 조성물을 제공한다.The extraction solvent provides a composition for improving bone metabolism, characterized in that at least one selected from water, a lower alcohol having 1 to 4 carbon atoms, a polyhydric alcohol, or a mixture thereof.
본 발명은, 다른 추가의 일면에 있어서,The present invention, in another further aspect,
상기 조성물은 식품 또는 의약 조성물인 것을 특징으로 하는 골 대사 개선용 조성물을 제공한다.The composition provides a composition for improving bone metabolism, characterized in that it is a food or pharmaceutical composition.
본 발명은, 다른 추가의 일면에 있어서,The present invention, in another further aspect,
상기 조성물은 골다공증 (Osteoporosis), 골연화증 (Osteomalacia), 골감소증 (Osteopenia), 골 위축(Bone atrophy), 섬유성 골이형성증 (Fibrous dysplasia), 파제트병 (Paget's disease), 고칼슘혈증(Hypercalcemia), 뼈의 종양성 파괴 (Neoplastic bone destruction), 및 골 성장장애 (Growth disorder of the bone)로 이루어진 군으로부터 선택되는 하나 이상의 질환을 개선하는 것을 특징으로 하는 골 대사 개선용 조성물을 제공한다.The composition comprises Osteoporosis, Osteomalacia, Osteopenia, Bone atrophy, Fibrous dysplasia, Paget's disease, Hypercalcemia, and bone It provides a composition for improving bone metabolism, characterized in that it improves at least one disease selected from the group consisting of neoplastic bone destruction, and growth disorder of the bone.
이하, 본 발명에 따른 골 대사 개선용 조성물을 첨부된 도면을 참고하여 더욱 상세하게 설명한다.Hereinafter, a composition for improving bone metabolism according to the present invention will be described in more detail with reference to the accompanying drawings.
표고버섯(Lentinus edodes)은 참나무 등 활엽수에 기생하는 담자균류 주름버섯목 느타리과에 속하며 향미 성분과 약리 효과를 가지고 있어 국내에서도 식용 및 약용으로 널리 이용되고 있다.Shiitake ( Lentinus edodes ) belongs to the family of Basidiomycete Pleurotus oleracea parasitic in broad-leaved trees such as oak, and has flavor components and pharmacological effects, so it is widely used for edible and medicinal purposes in Korea.
또한, 표고버섯은 식품으로서 뿐만 아니라 강장, 이뇨, 고혈압, 신장염, 신경쇠약, 불면증, 천식, 위궤양 등의 치료에 효능이 있는 것으로 알려져 있다.In addition, shiitake mushrooms are known to be effective in treating tonic, diuretic, hypertension, nephritis, nervous breakdown, insomnia, asthma, gastric ulcers, as well as as food.
상기 표고버섯은 깨끗이 세척하여 건조하여 세절한 다음 원료 중량의 5배 내지 20배의 추출용매를 더하여 고압멸균솥에 넣고 65 ~ 125℃의 온도에서 2 내지 72시간, 바람직하게는 5시간 동안 가열시켜 추출하여 여과하여 얻을 수 있다.The shiitake mushrooms are cleaned, dried, and minced, and then put in an autoclave by adding an extraction solvent of 5 to 20 times the weight of the raw material and heated at a temperature of 65 to 125°C for 2 to 72 hours, preferably 5 hours. It can be obtained by extraction and filtration.
여기서 추출용매로 물, 탄소수 1 내지 4의 저급 알코올, 다가 알코올 또는 이들의 혼합물로부터 선택된 적어도 어느 하나를 이용할 수 있다. 탄소수 1 내지 4의 저급 알코올로 메탄올, 에탄올 등을 이용할 수 있고, 다가 알코올로 부틸렌글리콜 및 프로필렌글리콜, 펜틸렌글리콜 등을 이용할 수 있다. 그리고 혼합물로는 물 및 저급 알코올의 혼합물, 물 및 다가 알코올의 혼합물, 저급 알코올 및 다가 알코올의 혼합물, 또는 물 및 저급알코올 및 다가알코올의 혼합물을 이용할 수 있다.Here, as the extraction solvent, at least one selected from water, a lower alcohol having 1 to 4 carbon atoms, a polyhydric alcohol, or a mixture thereof may be used. Methanol, ethanol, etc. can be used as the lower alcohol having 1 to 4 carbon atoms, and butylene glycol, propylene glycol, pentylene glycol, and the like can be used as the polyhydric alcohol. And as the mixture, a mixture of water and a lower alcohol, a mixture of water and a polyhydric alcohol, a mixture of a lower alcohol and a polyhydric alcohol, or a mixture of water and a lower alcohol and a polyhydric alcohol may be used.
별법으로, 상기 표고버섯은 추출용매를 가하여 열수추출, 냉침 또는 온침 추출할 수 있다. 이 경우 식물 재료에 대하여 추출용매를 중량비로 5 내지 20배를 가하여 혼합한 후 15 내지 125℃에서 2시간 내지 6개월 동안 추출하고, 여과하여 추출물을 얻을 수 있다.Alternatively, the shiitake mushrooms can be extracted with hot water, cold or warm needles by adding an extraction solvent. In this case, after mixing by adding an
본 발명에 의한 표고버섯 추출물은 후술하는 표고버섯 추출물의 골 조직 내 in vitro 효능 분석 및 표고버섯 추출물의 미네랄 형성 촉진 효과 검증을 통하여 시험 결과로부터 확인되는 바와 같이 골다공증(Osteoporosis), 골연화증 (Osteomalacia), 골감소증(Osteopenia), 골 위축(Bone atrophy), 섬유성 골이형성증(Fibrous dysplasia), 파제트병(Paget's disease), 고칼슘혈증(Hypercalcemia), 뼈의 종양성 파괴(Neoplastic bone destruction), 및 골 성장장애(Growth disorder of the bone)로 이루어진 군으로부터 선택되는 하나 이상의 질환을 개선하기 위한 의약 또는 건강 기능 제품의 유효 성분으로 활용이 가능할 것으로 판단된다.Shiitake mushroom extract according to the present invention is as confirmed from the test results through the in vitro efficacy analysis of the shiitake mushroom extract in bone tissue and the verification of the mineral formation promoting effect of the shiitake mushroom extract described below, osteoporosis, Osteomalacia, Osteopenia, bone atrophy, fibrous dysplasia, Paget's disease, hypercalcemia, neoplastic bone destruction, and bone growth disorders (Growth disorder of the bone), it is considered that it can be used as an active ingredient in medicine or health function products to improve one or more diseases selected from the group consisting of.
본 발명의 의약은 다양한 경로로 투여될 수 있다. 다양한 투여 방식이 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 또는 피하 주사에 의해 투여될 수 있다.The medicament of the present invention can be administered by various routes. Various modes of administration can be envisaged, for example oral, rectal or intravenous, intramuscular, or subcutaneous injection.
또한, 상기 의약 조성물은 실제 임상투여 시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있으며, 제제화할 경우에는 보통 사용하는 충진제, 중량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용할 수 있다. In addition, the pharmaceutical composition can be administered in various oral and parenteral formulations at the time of actual clinical administration, and when formulated, diluents or excipients such as commonly used fillers, weight agents, binders, wetting agents, disintegrants, surfactants, etc. You can use
경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include, for example, starch, calcium carbonate, sucrose or lactose, It is prepared by mixing gelatin or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. .
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used as the non-aqueous solvent and the suspension.
본 발명의 복합 추출물 함유 조성물의 투여 용량은 환자의 체충, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도에 따라 달라질 수 있으며, 의사 또는 약사의 판단에 따라 일정 시간 간격으로 1일 1회 내지 수회로 분할 투여할 수도 있다. 예컨대, 유효성분 함량 기준으로 1일 투여량이 0.001 내지 500㎎/㎏, 바람직하게는 0.1 내지 200㎎/㎏일 수 있다.The dosage of the composition containing the complex extract of the present invention may vary depending on the patient's body worms, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease, and is fixed according to the judgment of a doctor or pharmacist. It may be administered in divided doses from once to several times a day at intervals of time. For example, the daily dosage may be 0.001 to 500 mg/kg, preferably 0.1 to 200 mg/kg based on the active ingredient content.
또한, 본 발명에 따른 기능성 식품 조성물은 당해 분야에서의 통상적인 방법에 따라, 예를 들면 분말, 과립, 환, 정제, 캡슐, 액상 또는 음료 형태를 포함하는 제형으로 제공할 수 있다.In addition, the functional food composition according to the present invention may be provided in a dosage form including, for example, powder, granule, pill, tablet, capsule, liquid or beverage form according to a conventional method in the art.
본 발명의 식품의 섭취량은 그 제제 형태, 투여 방법, 사용 목적 및 이것에 적용되는 개체의 연령, 체중, 증상에 따라서 적절히 설정되고, 일정하지 않지만 일반적으로는 제제 중에 함유되는 유효성분의 양은 성인 1일당, 예컨대 0.001 mg/㎏∼500 ㎎/㎏이다. 물론 복용량은, 각종 조건에 의해서 변동하기 때문에, 상기 섭취량보다 적은 양으로 충분한 경우도 있고, 또는 범위를 초과하여 필요한 경우도 있다.The intake amount of the food of the present invention is appropriately set according to the formulation form, administration method, purpose of use, and the age, weight, and symptoms of the individual to which it is applied, and is not constant, but generally the amount of the active ingredient contained in the formulation is
또한, 본 발명의 조성물이 기능성 식품(건강기능식품 및 일반 식품) 조성물로 제조되는 경우에는 식품에 통상적으로 첨가되는 성분을 포함할 수 있으며, 예를 들어, 단백질, 탄수화물, 지방, 감미료, 예를 들면 감초, 비타민 C, 구연산, 니코틴산, 안식향산나트륨, 아스파탐, 사카린, 펙틴, 말리톨, 솔비톨, 자일리톨, 구아검, 탈지분유 및 올리고당으로 이루어진 군 중에서 선택되는 하나 이상의 성분을 추가하여 기호도나 미감을 증대시킬 수 있다.In addition, when the composition of the present invention is prepared as a functional food (health functional food and general food) composition, it may include ingredients that are commonly added to food, for example, protein, carbohydrate, fat, sweetener, for example For example, by adding one or more components selected from the group consisting of licorice, vitamin C, citric acid, nicotinic acid, sodium benzoate, aspartame, saccharin, pectin, malitol, sorbitol, xylitol, guar gum, skim milk powder, and oligosaccharides to increase preference or taste. I can make it.
이들은 본 발명의 조성물의 전체 중량을 기준으로 약 0.01~20 중량%로 사용하는 것이 적절하다.It is appropriate to use them in an amount of about 0.01 to 20% by weight based on the total weight of the composition of the present invention.
음료수는, 예를 들면, 상기 복합 추출물을 0.01~20 중량%으로 넣고, 감초 0.01~2 중량%, 구연산을 0.01~2 중량%, 사과산 0.01~2 중량%, 타우린 0.1∼2 중량%, 비타민 C 0.01~2 중량%, 비타민 B1 0.01~2 중량%, 바이오틴 0.01~2 중량%, 액상과당 0.01~20 중량% 폴리덱스트로스 0.1~3 중량%, 배농축액 0.1~1 중량%, 벌꿀 0.1~0.5중량%, 젖산칼슘 0.01~1 중량%, 향료 0.1~1 중량%, 색소 0.01~0.05 중량%, 구연산나트륨 0.01~0.5중량%, 스테비텐후레쉬 0.01~0.5 중량%, 니코틴산아미드 0.01~0.5 중량%, 로얄제리추출물 0.01~0.5 중량%, L-멘톨 0.0001~0.5 중량%등을 단독 또는 혼합하여 첨가하여 기능성 음료수로 제조할 수 있다.Drinking water, for example, put the complex extract in 0.01 to 20% by weight, licorice 0.01 to 2% by weight, citric acid 0.01 to 2% by weight, malic acid 0.01 to 2% by weight, taurine 0.1 to 2% by weight, vitamin C 0.01 to 2 wt%, vitamin B1 0.01 to 2 wt%, biotin 0.01 to 2 wt%, liquid fructose 0.01 to 20 wt%, polydextrose 0.1 to 3 wt%, pear concentrate 0.1 to 1 wt%, honey 0.1 to 0.5 wt%, Calcium lactate 0.01 to 1% by weight, fragrance 0.1 to 1% by weight, pigment 0.01 to 0.05% by weight, sodium citrate 0.01 to 0.5% by weight, stebiten fresh 0.01 to 0.5% by weight, nicotinic acid amide 0.01 to 0.5% by weight, royal jelly extract It can be prepared as a functional beverage by adding 0.01 to 0.5% by weight and 0.0001 to 0.5% by weight of L-menthol alone or as a mixture.
<실시예><Example>
이하, 본 발명은 다음의 대표적인 실시예에 의하여 더욱 구체적으로 설명되나, 본 발명이 이들 실시예에 의해 어떤 식으로든 제한되는 것은 아니다. 각 값은 3회 반복 시험한 것을 평균 ± 표준 편차로 나타내었다.Hereinafter, the present invention will be described more specifically by the following representative examples, but the present invention is not limited in any way by these examples. Each value is expressed as the mean ± standard deviation of the test repeated three times.
실시예Example 1: 표고버섯1: Shiitake mushroom 추출물의 제조 Preparation of extract
표고버섯 10 Kg를 깨끗이 세척하여 건조하여 세절한 다음 원료 중량의 10배의 정제수를 더하여 고압멸균솥에 넣고 100℃의 온도에서 5시간 동안 가열시켜 추출하여 여과하고 농축하여 추출물을 얻었다. 표고버섯의 추출 수율은 23.8%이고, brix는 3.4이었다.10 Kg of shiitake mushrooms were washed, dried, and minced. Then, 10 times the weight of the raw material was added to an autoclave, heated at 100° C. for 5 hours, extracted, filtered, and concentrated to obtain an extract. The extraction yield of shiitake mushrooms was 23.8%, and brix was 3.4.
실시예Example 2: 표고버섯 주정 추출물의 제조 2: Preparation of Shiitake Mushroom Alcohol Extract
1 Ton 추출기를 사용하여 표고버섯 200 Kg을 세척한 후, 세척된 표고버섯을 60℃의 온도로 열풍 건조시겼다. 건조된 표고버섯에 중량을 기준으로 20 배수의 주정을 가하여 0.5 기압 및 65℃의 온도에서 4시간 동안 추출하여 추출물을 얻었다. 이어서, 추출물을 여과지로 여과하여 이물질을 걸러낸 후, 여액을 60℃의 온도에서 -0.08 MPA의 감압하에서 8시간 동안 농축하였다. 고형분의 brix는 43으로 측정되었다. 이어서, 농축물을 진공하에서 60℃의 온도에서 24시간 동안 건조시키고, 이를 핀밀로 100 메쉬로 분쇄한 후 일정 단위로 소분하였다.After washing 200 Kg of shiitake mushrooms using a 1 Ton extractor, the washed shiitake mushrooms were dried with hot air at a temperature of 60°C. To the dried shiitake mushrooms, 20 folds of alcohol was added to the dried shiitake mushrooms and extracted for 4 hours at a temperature of 0.5 atm and 65°C to obtain an extract. Subsequently, the extract was filtered through a filter paper to filter out foreign matter, and the filtrate was concentrated for 8 hours under reduced pressure of -0.08 MPA at a temperature of 60°C. The brix of the solid was measured as 43. Then, the concentrate was dried under vacuum at a temperature of 60° C. for 24 hours, pulverized into 100 mesh with a pin mill, and then divided into predetermined units.
실시예Example 3: 정제의 제조 3: Preparation of tablets
실시예 3의 건조 추출 분말 50mg50 mg of dry extract powder of Example 3
옥수수전분 100 mg100 mg corn starch
유당 100 mg100 mg lactose
스테아린산 마그네슘 2 mg2 mg of magnesium stearate
정제 당 상기의 성분이 포함되도록 혼합한 후, 통상의 정제의 제조 방법에 따라서 타정하여 정제를 제조하였다.After mixing so as to contain the above components per tablet, tablets were prepared by tableting according to a conventional tablet preparation method.
실시예Example 4: 음료의 제조 4: Preparation of beverage
실시예 1의 건조 추출 분말 50mg50 mg of dry extract powder of Example 1
구연산 20mgCitric acid 20mg
올리고당 10mg10mg oligosaccharide
타우린 20mg20mg taurine
정제수를 가하여 전체 100 mlTotal 100 ml with purified water
통상의 건강 음료 제조방법에 따라 포장 용기 당 상기 함량을 포함하도록 음료를 제조하였다.Beverages were prepared to contain the above content per packaging container according to a conventional healthy beverage manufacturing method.
시험예Test example 1: 표고버섯 추출물의 알칼리 포스파타아제 활성 분석 1: Analysis of alkaline phosphatase activity of shiitake mushroom extract
골형성 지표로 alkaline phosphatase와 특이적 기질을 이용하여 효소유사 활성을 분석할 수 있다. 혈청 total alkaline phosphatase는 골아세포에서 분비되는 효소로 조골세포의 활성을 반영하고 그 중 일부는 뼈에 존재하여 골형성 정도를 반영해주는 지표이다. 따라서, 소재가 포함하고 있는 alkaline phosphatase 유사활성을 확인하고 활성 변화분석을 통해 소재의 alkaline phosphatase 유사활성 효과를 평가할 수 있다.The enzyme-like activity can be analyzed using alkaline phosphatase and a specific substrate as an index of bone formation. Serum total alkaline phosphatase is an enzyme secreted from osteoblasts and reflects the activity of osteoblasts, and some of them are present in bones, which reflects the degree of bone formation. Therefore, it is possible to check the alkaline phosphatase-like activity contained in the material and evaluate the effect of the alkaline phosphatase-like activity of the material through the activity change analysis.
실시예 1의 표고버섯 열수 추출물에 대하여 p-nitrophenyl phosphate liquid substrate system을 이용하여 spectrophotometric assay 수행하였다. 기질 용액에 다양한 농도의 시료를 상온에서 15분 반응시킨 후 405 nm에서 p-nitrophenol(PNP)를 측정하여 분석하였다. 또한, alkaline phosphatease 표준 효소를 이용하여 농도별 표준 값으로 시료의 alkaline phosphatase 유사활성을 분석하고, Microplate reader (Molecular Devices, Sunnyvale, CA, USA)를 이용하여 측정하였다.A spectrophotometric assay was performed on the shiitake mushroom hot water extract of Example 1 using a p-nitrophenyl phosphate liquid substrate system. Samples of various concentrations were reacted with the substrate solution at room temperature for 15 minutes, and then p-nitrophenol (PNP) was measured and analyzed at 405 nm. In addition, alkaline phosphatase-like activity of the sample was analyzed at standard values for each concentration using an alkaline phosphatease standard enzyme, and measured using a Microplate reader (Molecular Devices, Sunnyvale, CA, USA).
실험군으로 표고버섯 열수 추출물을 처리하였고 비교 분석을 위해 음성대조군은 무처리군, 양성대조군은 대조군은 0.2 mU의 alkaline phosphatase를 처리하였다. alkaline phosphatase와 시료의 처리시간은 10분이며 효소 특이적 합성기질의 처리시간은 30분으로 하였다. 표고버섯 열수 추출물의 alkaline phosphatase 활성을 측정하기 위해 먼저 0, 100, 200, 300, 400 micro unit (μU)의 효소와 효소 특이적 합성기질을 이용하여 표준 검정선을 만들어 이를 활용한 alkaline phosphatase 유사활성 평가를 수행하였다. 도 1은 특이 기질에 대한 알칼리 포스파타아제의 표준 그래프이다.The experimental group was treated with hot water extract of shiitake mushrooms, and for comparative analysis, the negative control group was treated with 0.2 mU alkaline phosphatase and the positive control group was treated with 0.2 mU alkaline phosphatase. The treatment time for alkaline phosphatase and the sample was 10 minutes, and the treatment time for the enzyme-specific synthetic substrate was 30 minutes. To measure the alkaline phosphatase activity of the hot water extract of shiitake mushrooms, a standard calibration line was first made using 0, 100, 200, 300, 400 micro unit (μU) of enzyme and enzyme-specific synthetic substrate, and the alkaline phosphatase-like activity using this Evaluation was performed. 1 is a standard graph of alkaline phosphatase for specific substrates.
도 2는 특이 기질에 대한 각 시료들의 평균 흡광도를 나타낸 그라프도이다. alkaline phosphatase 효소 특이적 기질에 각 시료처리 후 405 nm에서 흡광도 수치를 확인한 결과, 무처리 음성대조군은 0, 0.2 mU의 alkaline phosphatase를 처리한 양성대조군은 0.161, 표고버섯 열수 추출물 1 μg의 처리는 0.0025, 2 μg은 0.0045, 5 μg은 0.0051, 10 μg은 0.0083, 20 μg은 0.0083, 50 μg은 0.0099의 흡광도 수치를 보였다. 또한, 표고버섯 열수 추출물이 alkaline phosphatase 활성에 미치는 영향을 확인하기 위해 alkaline phosphatase와 표고버섯 열수 추출물 1 μg를 처리한 실험군에서는 0.0135, 2 μg은 0.0159, 5 μg은 0.0255, 10 μg은 0.0195, 20 μg은 0.018, 50 μg은 0.0189로 확인되었다.2 is a graph showing the average absorbance of each sample for a specific substrate. As a result of checking the absorbance at 405 nm after each sample treatment on an alkaline phosphatase enzyme-specific substrate, the negative control was 0.161 for the positive control treated with 0, 0.2 mU alkaline phosphatase, and the treatment with 1 μg of shiitake hot water extract was 0.0025. , 2 μg was 0.0045, 5 μg was 0.0051, 10 μg was 0.0083, 20 μg was 0.0083, and 50 μg was 0.0099. In addition, in order to confirm the effect of hot water extract of shiitake mushrooms on alkaline phosphatase activity, 0.0135, 2 μg of 0.0159, 5 μg of 0.0255, 10 μg of 0.0195, 20 μg in the experimental group treated with alkaline phosphatase and 1 μg of shiitake mushroom hot water extract Silver was 0.018 and 50 μg was confirmed to be 0.0189.
도 3은 각 시료의 평균 알칼리 포스파타아제 단위이다. 위 흡광도 수치로부터 alkaline phosphatase의 mU으로 환산하면 표고버섯 열수 추출물 1 μg의 처리는 0.03 mU, 2 μg은 0.06 mU, 5 μg은 0.06 mU, 10 μg은 0.10 mU, 20 μg은 0.10 mU, 50 μg은 0.12 mU의 효소유사 활성을 보였고, alkaline phosphatase에 표고버섯 열수 추출물을 처리한 시험에서는 표고버섯 열수 추출물 1 μg의 처리는 0.17 mU, 2 μg은 0.20 mU, 5 μg은 0.32 mU, 10 μg은 0.24 mU, 20 μg은 0.22 mU, 50 μg은 0.23 mU으로 확인되었다. 3 is an average alkaline phosphatase unit of each sample. Converting from the above absorbance to mU of alkaline phosphatase, treatment with 1 μg of shiitake mushroom hot water extract is 0.03 mU, 2 μg is 0.06 mU, 5 μg is 0.06 mU, 10 μg is 0.10 mU, 20 μg is 0.10 mU, and 50 μg is The enzyme-like activity of 0.12 mU was shown, and in the test in which the hot water extract of shiitake mushroom was treated with alkaline phosphatase, treatment with 1 μg of shiitake mushroom hot water extract was 0.17 mU, 0.20 mU for 2 μg, 0.32 mU for 5 μg, and 0.24 mU for 10 μg. , 20 μg was found to be 0.22 mU, and 50 μg was found to be 0.23 mU.
표고버섯 열수 추출물 50 μg에서 alkaline phosphatase의 0.2 mU에 해당하는 효소유사 활성을 보였고, 표고버섯 열수 추출물은 1-2 μg의 농도에서 alkaline phosphatase의 활성을 다소 감소시켰지만 5-50 μg의 농도에서는 alkaline phosphatase의 활성을 12-58 % 증가시켰다.In 50 μg of hot water extract of shiitake mushroom, enzyme-like activity equivalent to 0.2 mU of alkaline phosphatase was shown, and hot water extract of shiitake mushroom slightly reduced the activity of alkaline phosphatase at a concentration of 1-2 μg, but alkaline phosphatase at concentration of 5-50 μg Increased the activity of 12-58%.
또한, 표고버섯 열수 추출물의 다양한 농도와 표고버섯 열수 추출물을 처리한 alkaline phosphatase을 해당 기질에 10분 동안 반응시켜 Vmax 측정을 통해 kinetic 분석을 수행하였다. 이러한 kinetic 분석은 흡광도 수치보다 더 민감하게 효소반응의 변화를 확인할 수 있다.In addition, kinetic analysis was performed by measuring Vmax by reacting various concentrations of the shiitake mushroom hot-water extract and alkaline phosphatase treated with the shiitake hot-water extract with the corresponding substrate for 10 minutes. Such kinetic analysis can confirm the change of the enzyme reaction more sensitively than the absorbance value.
도 4는 특이 기질에 대한 시료의 평균 Vmax 값을 나타내는 그라프도이다. Vmax 측정결과, 0.2 mU의 alkaline phosphatase를 처리한 양성대조군은 0.1 mU/min, 표고버섯 열수 추출물 1 μg의 처리는 -1.43 mU/min, 2 μg은 -1.0 mU/min, 5 μg은 -1.89 mU/min, 10 μg은 -1.28 mU/min, 20 μg은 -0.43 mU/min, 50 μg은 -0.98 mU/min으로 확인되었다.4 is a graph showing the average Vmax value of a sample for a specific substrate. As a result of Vmax measurement, 0.1 mU/min for the positive control treated with 0.2 mU alkaline phosphatase, -1.43 mU/min for 1 μg of shiitake mushroom hot water extract, -1.0 mU/min for 2 μg, -1.89 mU for 5 μg /min, 10 μg was -1.28 mU/min, 20 μg was -0.43 mU/min, and 50 μg was -0.98 mU/min.
Alkaline phosphatase에 표고버섯 열수 추출물을 처리한 결과, 표고버섯 열수 추출물 1 μg의 처리는 1.31 mU/min, 2 μg은 2.98 mU/min, 5 μg은 5.63 mU/min, 10 μg은 2.95 mU/min, 20 μg은 6.57 mU/min, 50 μg은 2.62 mU/min으로 확인되었다. 아무것도 처리하지 않은 0.2 mU alkaline phosphatase의 Vmax를 100 %로 하여 비교분석한 결과, 표고버섯 열수 추출물 1 μg의 처리는 -35.8 %, 2 μg은 -25.0 %, 5 μg은 -47.3 %, 10 μg은 -31.9 %, 20 μg은 -10.8 %, 50 μg은 -24.6 %로 분석되었고 0.2 mU alkaline phosphatase의 Vmax는 표고버섯 열수 추출물 1 μg의 처리에 의해 32.7 %, 2 μg의 처리에 의해 74.6 %, 5 μg의 처리에 의해 140.8 %, 10 μg의 처리에 의해 73.8 %, 20 μg의 처리에 의해 164.2 %, 50 μg의 처리에 의해 65.4 %로 확인되었다.As a result of treatment with hot water extract of shiitake mushrooms in Alkaline phosphatase, treatment of 1 μg of shiitake mushroom hot water extract was 1.31 mU/min, 2 μg was 2.98 mU/min, 5 μg was 5.63 mU/min, 10 μg was 2.95 mU/min, 20 μg was found to be 6.57 mU/min and 50 μg was found to be 2.62 mU/min. As a result of comparative analysis using the Vmax of 0.2 mU alkaline phosphatase without treatment as 100%, treatment with 1 μg of shiitake mushroom hot water extract was -35.8%, 2 μg was -25.0%, 5 μg was -47.3%, and 10 μg was -31.9%, 20 μg were analyzed as -10.8%, and 50 μg as -24.6%. The Vmax of 0.2 mU alkaline phosphatase was 32.7% by treatment with 1 μg of shiitake mushroom hot water extract, 74.6% by treatment with 2 μg, 5 It was found to be 140.8% by treatment with μg, 73.8% by treatment with 10 μg, 164.2% by treatment with 20 μg, and 65.4% by treatment with 50 μg.
본 결과를 통해, 표고버섯 열수 추출물이 alkaline phosphatase 유사활성 효과를 가지며, 5 μg 농도 이상에서 alkaline phosphatase의 활성을 증가시켜 골형성 지표를 개선할 수 있을 것으로 기대된다.From these results, it is expected that the hot water extract of shiitake mushrooms has an alkaline phosphatase-like activity, and increases the activity of alkaline phosphatase at a concentration of 5 μg or more to improve bone formation index.
시험예Test example 2: 칼슘 성분 함량 분석 2: Analysis of calcium content
골 대사 균형이 무너지고 칼슘 대사가 손상되면 칼슘과 인의 농도가 감소하게 되므로, 칼슘 변화 분석은 골형성 지표로 활용할 수 있다. 칼슘 농도 측정은 OCPC법을 사용하였고 calcium kit의 측정시약(calcium kit; Bio clinical system corporation)을 사용하여 완충액 5 mL에 정색 시약 0.5 mL를 혼합하고, 시료 0.05 mL를 충분히 혼합한 후 실온에서 5 분간 방치한 다음 90분 이내에 파장 575 nm 흡광도를 측정하였다. calcium 표준 물질을 이용하여 농도별 표준 값으로 시료의 농도 분석하고, Microplate reader (Molecular Devices, Sunnyvale, CA, USA)를 이용하여 측정하였다.When the balance of bone metabolism is broken and calcium metabolism is impaired, the concentration of calcium and phosphorus decreases, so calcium change analysis can be used as an index of bone formation. The OCPC method was used to measure the calcium concentration, and 0.5 mL of the color reagent was mixed with 5 mL of the buffer using a calcium kit (Bio clinical system corporation), and 0.05 mL of the sample was sufficiently mixed, and then at room temperature for 5 minutes. After standing, the absorbance at 575 nm was measured within 90 minutes. The concentration of the sample was analyzed with standard values for each concentration using a calcium standard material, and measured using a Microplate reader (Molecular Devices, Sunnyvale, CA, USA).
실험군으로 표고버섯 열수 추출물을 처리하였고 비교분석을 위해 음성대조군은 무처리군, 실험군은 표고버섯 열수 추출물을 농도별로 10 μg, 20 μg, 50 μg, 100 μg의 칼슘 함량을 측정하였다.The experimental group was treated with hot water extract of shiitake mushrooms, and for comparative analysis, the negative control group was untreated and the experimental group measured the calcium content of 10 μg, 20 μg, 50 μg, and 100 μg of shiitake hot water extract by concentration.
도 5는 표고버섯 열수 추출물의 평균 칼슘 레벨을 나타내는 그라프도이다. 그 결과, 표고버섯 열수 추출물 10 μg에는 0.41 mg/dL의 칼슘을 함유하는 것으로 나타났고 20 μg에는 0.65 mg/dL의 칼슘, 50 μg에는 0.97 mg/dL의 칼슘, 100 μg에는 0.51 mg/dL의 칼슘을 함유하였다. 표고버섯 열수 추출물 50 μg에서 가장 높은 칼슘 함유량을 보였다.5 is a graph showing the average calcium level of hot water extract of shiitake mushrooms. As a result, it was found that 10 μg of shiitake mushroom hot water extract contained 0.41 mg/dL of calcium, 0.65 mg/dL of calcium in 20 μg, 0.97 mg/dL of calcium in 50 μg, and 0.51 mg/dL of calcium in 100 μg. Contains calcium. The highest calcium content was found in 50 μg of shiitake mushroom hot water extract.
시험예Test example 3: 3: cathepsincathepsin K 활성 K active 억제능Inhibitory ability 분석 analysis
파골세포는 골조직의 파괴, 재흡수를 담당하고 있고 골 흡수시 cathepsin K를 분비하여 제1형 콜라켄의 분해를 촉매하는 것으로 알려져 있다. 따라서, cathepsin K 활성억제는 골조직 파괴 및 흡수를 지연시켜 골형성에 변화를 줄 수 있기 때문에 cathepsin K 활성 변화는 골형성 지표로 활용할 수 있다.Osteoclasts are responsible for destruction and resorption of bone tissue, and are known to catalyze the decomposition of
Cathepsin K에 특이적인 기질 Leu-pNA chromogenic substrate를 이용하여 spectrophotometric assay 수행하고, 기질 용액에 다양한 농도의 시료와 효소를 상온에서 10-30분 반응시킨 후 405 nm에서 p-nitrophenol (PNP)를 Microplate reader (Molecular Devices, Sunnyvale, CA, USA)를 이용하여 측정하여 분석하였다.A spectrophotometric assay was performed using the Cathepsin K-specific substrate Leu-pNA chromogenic substrate, and after 10-30 minutes reaction of samples and enzymes of various concentrations to the substrate solution at room temperature, p-nitrophenol (PNP) was added at 405 nm to a Microplate reader. (Molecular Devices, Sunnyvale, CA, USA) was measured and analyzed.
효소활성 억제능은 다음과 같은 식을 이용하여 산출하였다. 억제 활성(%) = [1-(시료 처리군의 흡광도/시료 무처리군의 흡광도)] x 100.The enzyme activity inhibitory ability was calculated using the following equation. Inhibitory activity (%) = [1-(absorbance of sample-treated group/absorbance of sample-free group)] x 100.
실험군으로 표고버섯 열수 추출물을 처리하였고 비교분석을 위해 음성대조군은 무처리군, 양성대조군은 대조군은 0.1 mU의 cathepsin k를 처리하였다. Cathepsin과 시료의 반응시간은 10분이며 효소 특이적 합성기질의 처리시간은 30분으로 하였다.The experimental group was treated with hot water extract of shiitake mushrooms, and for comparative analysis, the negative control group was treated with 0.1 mU cathepsin k and the positive control group was treated with 0.1 mU of cathepsin k. The reaction time between Cathepsin and the sample was 10 minutes, and the treatment time of the enzyme-specific synthetic substrate was 30 minutes.
도 6은 시료의 특이 기질에 대한 평균 흡광도를 나타내는 그라프도이다. Cathepsin 효소 특이적 기질에 각 시료처리 후 405 nm에서 흡광도 수치를 확인한 결과, 무처리 음성대조군은 0, 1 mU의 cathepsin을 처리한 양성대조군은 0.0023, 표고버섯 열수 추출물 1 μg의 처리는 0.0086, 2 μg은 0.0092, 5 μg은 0.0136의 흡광도 수치를 보였다. 또한, 표고버섯 열수 추출물이 cathepsin 활성에 미치는 영향을 확인하기 위해 cathepsin과 표고버섯 열수 추출물 1 μg를 처리한 실험군에서는 0.0059, 2 μg은 0.0097, 5 μg은 0.0151로 확인되었다.6 is a graph showing the average absorbance for a specific substrate of a sample. As a result of confirming the absorbance values at 405 nm after each sample treatment on the Cathepsin enzyme-specific substrate, the negative control was 0.0023 for the positive control treated with 0, 1 mU of cathepsin, and 0.0086 and 2 for the treatment with 1 μg of shiitake hot water extract. The absorbance values were 0.0092 for μg and 0.0136 for 5 μg. In addition, in the experimental group treated with cathepsin and 1 μg of shiitake mushroom hot water extract to confirm the effect of shiitake mushroom hot water extract on cathepsin activity, it was found that 0.0059, 2 μg was 0.0097, and 5 μg was 0.0151.
도 7은 시료의 특이 기질에 대한 평균 카텝신 환산 단위를 나타내는 그라프도이다. 위 흡광도 수치를 가지고 cathepsin의 mU으로 환산하면 표고버섯 열수 추출물 1 μg의 처리는 3.74 mU, 2 μg은 4.00 mU, 5 μg은 5.91 mU의 효소유사 활성을 보였으며, cathepsin에 표고버섯 열수 추출물을 처리한 시험에서는 표고버섯 열수 추출물 1 μg의 처리는 2.57 mU, 2 μg은 4.22 mU, 5 μg은 6.57 mU으로 확인되었다.7 is a graph showing average cathepsin conversion units for specific substrates of a sample. With the above absorbance values and converted into mU of cathepsin, treatment of 1 μg of shiitake mushroom hot water extract showed an enzyme-like activity of 3.74 mU, 2 μg of 4.00 mU, and 5 μg of 5.91 mU. Cathepsin was treated with shiitake hot water extract. In one test, treatment with 1 μg of shiitake mushroom hot water extract was 2.57 mU, 2 μg was 4.22 mU, and 5 μg was 6.57 mU.
결과적으로, 표고버섯 열수 추출물은 4-6 mU에 해당하는 효소유사활성을 보였고, 표고버섯 열수 추출물은 모든 농도에서 cathepsin의 활성을 다소 증가시켰다.As a result, hot water extract of shiitake mushroom showed enzyme-like activity corresponding to 4-6 mU, and hot water extract of shiitake mushroom slightly increased cathepsin activity at all concentrations.
또한, 표고버섯 열수 추출물의 다양한 농도와 표고버섯 열수 추출물을 처리한 cathepsin을 해당 기질에 10분 동안 반응시켜 Vmax 측정을 통해 kinetic 분석을 수행하였다. In addition, kinetic analysis was performed by measuring Vmax by reacting various concentrations of shiitake mushroom hot-water extract and cathepsin treated with shiitake hot-water extract to the corresponding substrate for 10 minutes.
도 8은 시료의 특이 기질에 대한 평균 Vmax 값을 나타내는 그라프도이다. Vmax 측정결과, 1 mU의 cathepsin을 처리한 양성대조군은 -0.158 mU/mg/min, 표고버섯 열수 추출물 1 μg의 처리는 -0.441 mU/mg/min, 2 μg은 -0.412 mU/mg/min, 5 μg은 -0.350 mU/mg/min으로 확인되었다. Cathepsin에 표고버섯 열수 추출물을 처리한 결과, 표고버섯 열수 추출물 1 μg의 처리는 -0.158 mU/mg/min, 2 μg은 -0.467 mU/mg/min, 5 μg은 -0.532 mU/mg/min으로 확인되었다.8 is a graph showing the average Vmax value for a specific substrate of a sample. As a result of Vmax measurement, the positive control treated with 1 mU of cathepsin was -0.158 mU/mg/min, the treatment with 1 μg of shiitake mushroom hot water extract was -0.441 mU/mg/min, 2 μg was -0.412 mU/mg/min, 5 μg was found to be -0.350 mU/mg/min. As a result of treatment with hot water extract of shiitake mushrooms in Cathepsin, treatment with 1 μg of shiitake mushroom hot water extract was -0.158 mU/mg/min, 2 μg was -0.467 mU/mg/min, and 5 μg was -0.532 mU/mg/min. Confirmed.
도 9는 시료의 평균 Vmax 값의 백분율을 나타내는 그라프도이다. 아무것도 처리하지 않은 1 mU cathepsin의 Vmax를 100%로 하여 비교분석한 결과, 표고버섯 열수 추출물 1 μg의 처리는 -369.1 %, 2 μg은 -338.3 %, 5 μg은 -272.3 %로 분석되었고 1 mU cathepsin의 Vmax는 표고버섯 열수 추출물 1 μg의 처리에 의해 -168.1 %, 2 μg의 처리에 의해 -496.8 %, 5 μg의 처리에 의해 -566.0 %로 확인되었다.9 is a graph showing the percentage of an average Vmax value of a sample. As a result of comparative analysis using the Vmax of 1 mU cathepsin without treatment as 100%, treatment with 1 μg of shiitake mushroom hot water extract was -369.1%, 2 μg was -338.3%, 5 μg was -272.3%, and 1 mU The Vmax of cathepsin was confirmed to be -168.1% by treatment with 1 μg of shiitake mushroom hot water extract, -496.8% by treatment with 2 μg, and -566.0% by treatment with 5 μg.
시험예Test example 4: 골 조직 내 NO 분석 4: Analysis of NO in bone tissue
일산화질소(NO)는 파골세포의 생장을 억제하고 Type IV collagenase/gelatinase의 활성을 감소시키는 역할을 하는 것으로 알려져 있다. 따라서, 골조직 내 NO 함량 변화 분석은 골 형성 지표로 활용할 수 있다.Nitric oxide (NO) is known to play a role in inhibiting the growth of osteoclasts and reducing the activity of Type IV collagenase/gelatinase. Therefore, analysis of changes in NO content in bone tissue can be used as an index for bone formation.
실험동물을 해부 전 24시간 절식시킨 후 ether 마취 하에서 개복한 후 대퇴골 골 조직을 분리하여 분석할 때 까지 -70℃에 보관하였다. 골 조직은 멸균생리수로 수 회 씻어낸 후 lysis buffer를 첨가하여 lysis 및 파쇄하여 원심분리(10000 x g) 후 골 조직 균질액을 조제하고, BCA kit로 단백질을 정량하였다. 시료는 적정 농도 (10 μg-1000 μg)를 산정하여 골 조직 균질액에 시료를 30분 또는 1시간 처리 후 여러 in vitro 시험을 수행하였다.The experimental animals were fasted for 24 hours before dissection, opened under ether anesthesia, and then the femur bone tissue was separated and stored at -70°C until analysis. Bone tissue was washed several times with sterile physiological water, lysis buffer was added, lysis and crushed, centrifugation (10000 x g), bone tissue homogenate was prepared, and protein was quantified with a BCA kit. For the sample, the appropriate concentration (10 μg-1000 μg) was calculated, and the sample was treated in a bone tissue homogenate for 30 minutes or 1 hour, followed by several in vitro tests.
시료 및 균질액의 최종부피를 0.4 mL로 하고 Griess reagent 0.2 mL를 가하여 잘 혼합한 다음, 실온에서 15분간 반응시키고 Microplate reader (Molecular Devices, Sunnyvale, CA, USA)를 이용하여 520 nm에서 흡광도를 측정하고 sodium nitrite의 농도별 표준값을 이용하여 잔존하는 nitrite의 양을 산출하였다. 대조군으로 증류수, 음성대조군으로 Griess reagent 대신 증류수 사용하였다.Make the final volume of the sample and homogenate 0.4 mL, add 0.2 mL of Griess reagent, mix well, react at room temperature for 15 minutes, and measure the absorbance at 520 nm using a Microplate reader (Molecular Devices, Sunnyvale, CA, USA) Then, the amount of remaining nitrite was calculated using the standard value for each concentration of sodium nitrite. Distilled water was used as a control, and distilled water was used instead of Griess reagent as a negative control.
실험군으로 골조직에 표고버섯 열수 추출물을 처리하였고 비교분석을 위해 음성대조군은 무처리군과 골조직만 반응시킨 것을 비교하였다. 표고버섯 열수 추출물을 농도별로 5 μg, 10 μg, 20 μg, 50 μg, 100 μg을 10분 동안 처리한 후 NO 함량을 측정하였다. NO함량은 nitrite 농도로 간접적 측정을 활용하였다.As the experimental group, the bone tissue was treated with hot water extract of shiitake mushrooms, and for comparative analysis, the negative control group was compared with that of the untreated group and only the bone tissue. Shiitake mushroom hot water extract was treated with 5 μg, 10 μg, 20 μg, 50 μg, and 100 μg for 10 minutes for each concentration, and then the NO content was measured. NO content was measured indirectly by nitrite concentration.
도 10은 조직 균질화물에 대한 열수 추출물의 NO 레벨을 나타내는 그라프도이다. 520 nm에서 흡광도를 측정한 결과, 추출물을 처리하지 않은 골조직에서는 0.0079, 표고버섯 열수 추출물 5 μg을 처리한 골조직에서는 0.0113, 10 μg을 처리한 골조직에서는 0.0128, 20 μg을 처리한 골조직에서는 0.0145, 50 μg을 처리한 골조직에서는 0.0176, 100 μg을 처리한 골조직에서는 0.0208의 흡광도 수치를 확인하였다. 이를 mM 농도로 환산한 결과, 추출물을 처리하지 않은 골조직에서는 0.16 mM, 표고버섯 열수 추출물 5 μg을 처리한 골조직에서는 0.20 mM, 10 μg을 처리한 골조직에서는 0.21 mM, 20 μg을 처리한 골조직에서는 0.23 mM, 50 μg을 처리한 골조직에서는 0.26 mM, 100 μg을 처리한 골조직에서는 0.29 mM의 NO 농도를 확인하였다. 10 is a graph showing the NO level of hot water extract for tissue homogenates. As a result of measuring the absorbance at 520 nm, 0.0079 in bone tissue without extract, 0.0113 in bone tissue treated with 5 μg shiitake mushroom hot water extract, 0.0128 in bone tissue treated with 10 μg, 0.0145 in bone tissue treated with 20 μg, 50 The absorbance values of 0.0176 in the bone tissue treated with μg and 0.0208 in the bone tissue treated with 100 μg were confirmed. As a result of converting this to mM concentration, 0.16 mM in bone tissue without extract, 0.20 mM in bone tissue treated with 5 μg shiitake mushroom hot water extract, 0.21 mM in bone tissue treated with 10 μg, 0.23 in bone tissue treated with 20 μg In the bone tissue treated with mM and 50 μg, the concentration of NO was 0.26 mM, and in the bone tissue treated with 100 μg, the NO concentration was 0.29 mM.
도 11은 조직 균질화물에 대한 열수 추출물의 NO 레벨의 백분율을 나타내는 그라프도이다. 골조직만 처리한 대조군의 0.16 mM 농도값을 100%로 하여 비교분석한 결과, 표고버섯 열수 추출물 5 μg을 처리한 골조직에서는 120.9 %, 10 μg을 처리한 골조직에서는 130.1 %, 20 μg을 처리한 골조직에서는 140.5 %, 50 μg을 처리한 골조직에서는 159.5 %, 100 μg을 처리한 골조직에서는 179.2 %로 나타났다. 추출물 농도가 증가할수록 NO 농도가 비례적으로 증가하였으며, 고농도에서 비교적 우수한 골조직 파괴 억제효과가 기대된다.11 is a graph showing the percentage of NO levels of hot water extract with respect to tissue homogenates. As a result of comparative analysis using the 0.16 mM concentration value of the control group treated with only bone tissue as 100%, 120.9% in bone tissue treated with 5 μg of shiitake mushroom hot water extract, 130.1% in bone tissue treated with 10 μg, and bone tissue treated with 20 μg In 140.5%, in bone tissue treated with 50 μg, 159.5%, and in bone tissue treated with 100 μg, 179.2%. As the extract concentration increased, the NO concentration was proportionally increased, and relatively excellent bone tissue destruction inhibitory effect was expected at high concentration.
시험예Test example 5: 골 조직 내 alkaline 5: alkaline in bone tissue phosphatasephosphatase 활성 분석 Activity assay
P-nitrophenyl phosphate liquid substrate system을 이용하여 spectrophotometric assay 수행하였다. 기질 용액에 다양한 농도의 시료가 처리된 균질액을 상온에서 15분 반응시킨 후 405 nm에서 p-nitrophenol(PNP)를 측정하여 분석하였다. alkaline phosphatease 표준 효소를 이용하여 농도별 표준 값으로 균질액의 alkaline phosphatase 활성 분석하고, Microplate reader (Molecular Devices, Sunnyvale, CA, USA)를 이용하여 측정하였다.Spectrophotometric assay was performed using a P-nitrophenyl phosphate liquid substrate system. After reacting the homogenate in which samples of various concentrations were treated in the substrate solution for 15 minutes at room temperature, p-nitrophenol (PNP) was measured and analyzed at 405 nm. The alkaline phosphatase activity of the homogenate was analyzed at standard values for each concentration using an alkaline phosphatease standard enzyme, and measured using a Microplate reader (Molecular Devices, Sunnyvale, CA, USA).
실험군으로 표고버섯 열수 추출물을 처리하였고 비교분석을 위해 음성대조군은 골조직에 아무것도 처리하지 않았고 양성대조군은 골조직에 표고버섯 열수 추출물을 농도별로 처리하였다. 골조직과 추출물의 반응은 10분 동안 하였고 반응 후 특이적 기질에서 30분 동안 반응시켜 405nm에서 흡광도를 측정하여 비교분석하였다.The experimental group was treated with hot water extract of shiitake mushrooms. For comparative analysis, the negative control group did not treat any bone tissue, and the positive control group treated the bone tissue with hot water extract of shiitake mushrooms by concentration. The reaction between the bone tissue and the extract was performed for 10 minutes, and after the reaction, the reaction was performed on a specific substrate for 30 minutes, and the absorbance was measured at 405 nm for comparative analysis.
도 12는 특이 기질에 대한 시료의 평균 흡광도를 나타내는 그라프도이다. Alkaline phosphatase 효소 특이적 기질에 각 시료처리 후 405 nm에서 흡광도 수치를 확인한 결과, 무처리 음성대조군은 0, 골조직만 처리된 반응액은 0.1214, 골조직에 1 mU의 alkaline phosphatase를 처리한 양성대조군은 0.1403, 표고버섯 열수 추출물 5 μg의 처리는 0.1189, 10 μg은 0.1242, 20 μg은 0.1241, 50 μg은 0.1267, 100 μg은 0.1264의 흡광도 수치를 보였다. 12 is a graph showing the average absorbance of a sample for a specific substrate. As a result of checking the absorbance level at 405 nm after each sample treatment on an alkaline phosphatase specific substrate, 0 for the untreated negative control, 0.1214 for the reaction solution treated with bone tissue only, and 0.1403 for the positive control treated with 1 mU alkaline phosphatase in bone tissue. , Treatment with 5 μg of shiitake mushroom hot water extract showed absorbance values of 0.1189, 10 μg 0.1242, 20 μg 0.1241, 50 μg 0.1267, and 100 μg 0.1264.
도 13은 특이 기질에 대한 시료의 평균 알칼리 포스파타아제 단위를 나타내는 그라프도이다. 위 흡광도 수치를 가지고 alkaline phosphatase의 mU으로 환산하면 골조직만 처리한 반응액은 0.87 mU, 표고버섯 열수 추출물 5 μg의 처리는 0.85 mU, 10 μg은 0.89 mU, 20 μg은 0.88 mU, 50 μg은 0.90 mU, 100 μg은 0.90 mU, 골조직에 1 mU의 alkaline phosphatase를 처리한 양성대조군은 1.0 mU의 효소유사활성 및 효소활성을 보였다.13 is a graph showing the average alkaline phosphatase units of a sample against a specific substrate. Using the above absorbance values and converting to mU of alkaline phosphatase, the reaction solution treated with only bone tissue is 0.87 mU, the treatment with 5 μg of shiitake mushroom hot water extract is 0.85 mU, 10 μg is 0.89 mU, 20 μg is 0.88 mU, and 50 μg is 0.90. The positive control group treated with alkaline phosphatase of 0.90 mU in mU and 100 μg and 1 mU in bone tissue showed enzyme-like and enzymatic activity of 1.0 mU.
골조직에 표고버섯 열수 추출물의 처리로 조직 내 alkaline phosphatase 활성이 증가하는 경향을 보였지만 유의미한 결과를 확인하지 못했다. 그러나, 표고버섯 열수 추출물은 골조직 내 alkaline phosphatase 활성을 약 3% 증가시키는 결과를 보였다.Alkaline phosphatase activity in tissues tended to increase by treatment with hot water extract of shiitake mushrooms on bone tissue, but no significant results were confirmed. However, the hot water extract of shiitake mushrooms showed a result of increasing alkaline phosphatase activity by about 3% in bone tissue.
시험예Test example 6: 골 조직 내 calcium 농도 분석 6: Analysis of calcium concentration in bone tissue
골 조직 내 칼슘농도 측정은 OCPC법을 사용하였고 calcium kit의 측정시약(calcium kit; Bio clinical system corporation)을 사용하여 완충액 5 mL에 정색 시약 0.5 mL를 혼합하고, 균질액 시료 0.05 mL를 충분히 혼합한 후 실온에서 5 분간 방치한 다음 90분 이내에 파장 575 nm 흡광도를 측정하였다. calcium 표준물질을 이용하여 농도별 표준값으로 시료의 농도 분석하고, Microplate reader (Molecular Devices, Sunnyvale, CA, USA)를 이용하여 측정하였다.The OCPC method was used to measure the calcium concentration in bone tissue, and 0.5 mL of the color reagent was mixed with 5 mL of the buffer using a calcium kit (Bio clinical system corporation), and 0.05 mL of the homogenate sample was sufficiently mixed. Then, it was allowed to stand at room temperature for 5 minutes, and then absorbance at a wavelength of 575 nm was measured within 90 minutes. The concentration of the sample was analyzed using a standard value for each concentration using a calcium standard material, and measured using a Microplate reader (Molecular Devices, Sunnyvale, CA, USA).
실험군으로 골조직에 표고버섯 열수 추출물을 처리하였고 비교분석을 위해 대조군은 골조직에 아무것도 처리하지 않았고, 실험군은 표고버섯 열수 추출물을 농도별로 10 μg, 20 μg, 50 μg, 100 μg을 10분 동안 반응시킨 후 칼슘 함량을 측정하였다.As the experimental group, the bone tissue was treated with hot water extract of shiitake mushrooms, and the control group did not treat any bone tissue for comparative analysis, and the experimental group reacted with 10 μg, 20 μg, 50 μg, and 100 μg of shiitake mushroom extract by concentration for 10 minutes. After the calcium content was measured.
도 14는 조직 균질화물에 대한 시료의 평균 칼슘 레벨을 나타내는 그라프도이다. 그 결과, 골조직만 처리한 대조군에서는 0.56 mg/dL의 칼슘을 함유하는 것으로 나타났고 골조직에 표고버섯 열수 추출물 10 μg을 처리한 반응액에서는 0.82 mg/dL, 20 μg에서는 0.87 mg/dL, 50 μg에는 0.51 mg/dL, 100 μg에는 0.73 mg/dL의 칼슘을 함유하였다. 표고버섯 열수 추출물 20 μg을 처리한 골조직에서 가장 높은 칼슘 함유량을 보였으며, 저농도의 추출물을 처리한 실험군에서 비교적 높은 조직 내 칼슘 함유량을 나타냈다.14 is a graph showing the average calcium level of a sample with respect to a tissue homogenate. As a result, it was found that the control group treated with only bone tissue contained 0.56 mg/dL of calcium, and 0.82 mg/dL in the reaction solution treated with 10 μg of shiitake mushroom hot water extract in the bone tissue, 0.87 mg/dL and 50 μg in 20 μg. 0.51 mg/dL of calcium and 0.73 mg/dL of calcium were contained in 100 μg. The highest calcium content was shown in bone tissue treated with 20 μg of shiitake mushroom hot water extract, and relatively high calcium content was found in the experimental group treated with the low concentration of extract.
시험예Test example 7: 골 조직 내 단백질 분석 7: Analysis of proteins in bone tissue
골 조직은 멸균생리수로 수 회 씻어낸 후 lysis buffer를 첨가하여 lysis 및 파쇄하여 원심분리(10000 x g) 후 골 조직 균질액을 조제하여 BCA kit로 단백질을 정량하였다. Bovine serum albumin을 표준 단백질로 이용하여 농도별 표준값으로 균질액의 단백질 농도를 Microplate reader (Molecular Devices, Sunnyvale, CA, USA)를 이용하여 562 nm에서 분석하였다.Bone tissue was washed several times with sterile physiological water, lysis buffer was added, lysis and crushed, centrifugation (10000 x g), bone tissue homogenate was prepared, and protein was quantified with a BCA kit. Using bovine serum albumin as a standard protein, the protein concentration of the homogenate as a standard value for each concentration was analyzed at 562 nm using a Microplate reader (Molecular Devices, Sunnyvale, CA, USA).
골조직 내 단백질 변화를 측정하고 골형성 단백질 및 골조직 단백질에 대한 골형성 지표를 평가하였다. 실험군으로 골조직에 표고버섯 열수 추출물을 처리하였고 비교분석을 위해 대조군은 골조직에 아무것도 처리하지 않았고, 실험군은 표고버섯 열수 추출물을 농도별로 5 μg, 10 μg, 20 μg, 50 μg, 100 μg을 10분 동안 반응시킨 후 단백질 함량을 측정하였다.Protein changes in bone tissue were measured, and bone formation indices for bone formation protein and bone tissue protein were evaluated. As the experimental group, the bone tissue was treated with hot water extract of shiitake mushrooms, and the control group did not treat any bone tissue for comparative analysis. After reacting during, the protein content was measured.
도 15는 조직 균질화물에 대한 열수 추출물의 평균 단백질 농도를 나타내는 그라프도이다. 그 결과, 골조직만 처리한 대조군에서는 7.605 mg/g의 단백질을 함유하는 것으로 나타났고 골조직에 표고버섯 열수 추출물 5 μg을 처리한 반응액에서는 8.787 mg/g, 10 μg에서는 9.013 mg/g, 20 μg에서는 8.470 mg/g, 50 μg에는 8.113 mg/g, 100 μg에는 0.73 mg/g의 단백질을 함유하였다.15 is a graph showing the average protein concentration of hot water extract for tissue homogenates. As a result, it was found that the control group treated with bone tissue only contained 7.605 mg/g protein, and 8.787 mg/g in the reaction solution treated with 5 μg of shiitake mushroom hot water extract in bone tissue, 9.013 mg/g and 20 μg in 10 μg The protein was 8.470 mg/g, 50 μg 8.113 mg/g, and 100 μg 0.73 mg/g.
도 16은 조직 균질화물에 대한 열수 추출물의 평균 단백질 농도의 백분율을 나타내는 그라프도이다. 골조직만 처리한 대조군의 7.605 mg/g 농도값을 100 %로 하여 비교분석한 결과, 표고버섯 열수 추출물 5 μg을 처리한 골조직에서는 115.5 %, 10 μg을 처리한 골조직에서는 118.5 %, 20 μg을 처리한 골조직에서는 111.4 %, 50 μg을 처리한 골조직에서는 113.1 %, 100 μg을 처리한 골조직에서는 106.7 %로 나타났다. 16 is a graph showing the percentage of the average protein concentration of the hot water extract with respect to the tissue homogenate. As a result of comparative analysis with the concentration value of 7.605 mg/g of the control group treated with only bone tissue as 100%, 115.5% in bone tissue treated with 5 μg of shiitake mushroom hot water extract, 118.5% in bone tissue treated with 10 μg, and 20 μg were treated. In one bone tissue, 111.4%, in bone tissue treated with 50 μg 113.1%, and in bone tissue treated with 100 μg were found to be 106.7%.
고농도 추출물에서 비교적 낮은 단백질 농도를 보였고 표고버섯 열수 추출물 20 μg을 처리한 골조직에서 가장 높은 단백질 농도를 보였으며, 저농도의 추출물을 처리한 실험군에서 비교적 높은 조직 내 단백질량을 나타냈다.The high-concentration extract showed relatively low protein concentration, the highest protein concentration in the bone tissue treated with 20 μg of shiitake mushroom hot water extract, and the relatively high amount of protein in the tissue in the experimental group treated with the low-concentration extract.
시험예Test example 8: 조골세포에 대한 세포 독성 및 세포 증식 효과 8: Cytotoxicity and cell proliferation effect on osteoblasts
시료의 농도별 처리에 따른 조골세포에 따른 독성 정도는 MC 3T3-E1 세포를 이용하여 MTT assay로 측정하였다. 조골세포를 96-well plate에 분주하여 2일간 배양한 후, 시료를 농도별로 처리하여 48시간 동안 배양하였다. 배양 후 MTT 시약을 5 mg/mL 농도로 20 μL를 각 well에 첨가하여 2시간 더 배양한 후 배지를 제거하고 DMSO를 200 μL씩 첨가하여 생성된 불용성의 formazan 결정을 용해시켜 ELISA reader로 570 nm에서 흡광도를 측정하였다. 세포의 증식률은 시료의 흡광도를 대조군의 흡광도에 대한 백분율로 나타낸다.The degree of toxicity according to osteoblasts according to the treatment of each concentration of the sample was measured by MTT assay using MC 3T3-E1 cells. After dispensing osteoblasts into a 96-well plate and culturing for 2 days, the samples were treated by concentration and cultured for 48 hours. After incubation, 20 μL of MTT reagent at a concentration of 5 mg/mL was added to each well and incubated for 2 more hours, and then the medium was removed, and 200 μL of DMSO was added to dissolve the insoluble formazan crystals generated by ELISA reader at 570 nm. Absorbance was measured at. The cell proliferation rate represents the absorbance of the sample as a percentage of the absorbance of the control group.
도 17은 MC 3T3-E1 세포주에 대한 표고버섯 추출물의 세포 생존능에 대한 영향을 나타내는 그라프도이고, 도 18은 MC 3T3-E1 세포주에 대한 비타민D-표고버섯 추출물의 세포 생존능에 대한 영향을 나타내는 그라프도이다. 조골세포에 10, 50, 100, 200, 300, 400, 500μg/ml농도로 처리한 후 표고버섯에서는 106, 99, 100, 99, 100, 93, 85%의 세포 생존율을 나타냈으며, 비타민 D-표고버섯에서는 100, 101, 99, 101, 103, 97, 83%로 세포 생존율을 나타냈다.17 is a graph showing the effect of shiitake mushroom extract on cell viability against MC 3T3-E1 cell line, and FIG. 18 is a graph showing the effect of vitamin D-shiitake mushroom extract on cell viability against MC 3T3-E1 cell line Is also. After treatment with osteoblasts at 10, 50, 100, 200, 300, 400, 500μg/ml concentration, shiitake mushrooms showed cell survival rates of 106, 99, 100, 99, 100, 93, 85%, and vitamin D- In shiitake mushrooms, cell viability was shown at 100, 101, 99, 101, 103, 97, 83%.
도 19는 MC 3T3-E1 세포주에 대한 표고버섯 추출물의 세포 증식에 대한 영향을 나타내는 그라프도이고, 도 20은 MC 3T3-E1 세포주에 대한 비타민D-표고버섯 추출물의 세포 증식에 대한 영향을 나타내는 그라프도이다. 24시간 동안 처리하여 확인한 결과를 바탕으로 세포 생존율 감소에 영향을 미치는 않았던 농도를 이용하여 조골세포의 증식에 미치는 영향을 알아보기 위해 72시간 동안 처리 후 측정하였다. 조골세포에 10, 50, 100, 200, 300 μg/ml 농도로 처리한 후 표고버섯에서는 103, 110, 124, 130% 로 세포 생존율을 나타냈으며, 비타민 D-표고버섯에서는 104, 119, 133, 145, 142%로 세포 생존율을 나타냈다. 19 is a graph showing the effect of shiitake mushroom extract on cell proliferation against MC 3T3-E1 cell line, and FIG. 20 is a graph showing the effect of vitamin D-shiitake mushroom extract on cell proliferation against MC 3T3-E1 cell line Is also. Based on the results confirmed by treatment for 24 hours, concentrations that did not affect the reduction in cell viability were measured after treatment for 72 hours to determine the effect on the proliferation of osteoblasts. After treatment with osteoblasts at concentrations of 10, 50, 100, 200, 300 μg/ml, the cell viability was 103, 110, 124, 130% in shiitake mushrooms, and 104, 119, 133, in vitamin D-shiitake mushrooms, Cell viability was shown at 145 and 142%.
시험예Test example 9: mineralization에 미치는 효과 분석 9: Analysis of effects on mineralization
조골세포를 2 x 104 cell/well 농도로 96-well plate에 분주한 후 세포가 90% 포화되게 자라면 10 mM β-glycerophosphate와 50 μg/mL ascorbic acid를 함유한 배지로 교환하여 2일마다 배지를 갈아주며 분화를 유도하였다.After dispensing osteoblasts into a 96-well plate at a concentration of 2 x 10 4 cells/well, if the cells grow to 90% saturation, exchange them with a medium containing 10 mM β-glycerophosphate and 50 μg/mL ascorbic acid every 2 days. Differentiation was induced by changing the medium.
조골세포 분화 10일 후 각 농도별 시료를 처리하여 48시간 배양하였다. 분석을 위해 배지를 제거하고 PBS로 세척한 뒤, 70% EtOH로 실온에서 1시간 동안 고정시켰다. 세포를 고정시킨 후 40 mM alizarine red(AR) solution으로 10분간 염색하고 3차 증류수로 세척한 뒤 10% cetylpyridinium chloride를 첨가하여 15분간 녹여 염색된 정도를 ELISA reader를 이용하여 561nm에서 흡광도를 측정하였다. 석회화(mineralization) 정도는 대조군에 대한 백분율로 나타냈다.After 10 days of osteoblast differentiation, samples for each concentration were treated and cultured for 48 hours. For analysis, the medium was removed, washed with PBS, and fixed with 70% EtOH at room temperature for 1 hour. After fixing the cells, stained with 40 mM alizarine red (AR) solution for 10 minutes, washed with 3rd distilled water, dissolved with 10% cetylpyridinium chloride for 15 minutes, and measured the absorbance at 561 nm using an ELISA reader. . The degree of mineralization was expressed as a percentage of the control group.
골석회화 형성능은 조골세포의 분화에 중요한 biomarker로 calcium에 특이적으로 흡착력이 높은 Alizarin을 이용하여 세포를 염색하게 되면 석회화된 양과 염색 정도가 상호 비례하게 된다. 추출물의 농도에 따른 석회화 형성도를 확인하기 위해 염색된 석회화물을 10% cetypyrinium chloride로 녹여 흡광도 값을 측정하여 석회화에 미치는 영향을 확인하였다. Osteocalcification formation ability is an important biomarker for the differentiation of osteoblasts. When cells are stained with Alizarin, which has a high adsorption capacity specifically for calcium, the amount of calcification and the degree of staining are mutually proportional. To determine the degree of calcification according to the concentration of the extract, the dyed calcification was dissolved with 10% cetypyrinium chloride, and the absorbance value was measured to confirm the effect on calcification.
도 21은 표고버섯 추출물의 MC3T3-E1 세포주에서 골석회화 형성능에 대한 영양을 나타내는 그라프도이고, 도 22는 비타민D-표고버섯 추출물의 MC3T3-E1 세포주에서 골석회화 형성능에 대한 영양을 나타내는 그라프도이다. 0, 10, 50, 100, 200μg/ml 농도로 14주일간 처리 후 염색하여 확인한 결과, 표고버섯에서는 99, 100, 112, 123% 로 골석회화 형성능을 나타냈으며, 비타민 D-표고버섯에서는 100, 108, 126, 137%로 골석회화 형성능을 나타냈다. FIG. 21 is a graph showing nutrition for bone calcification formation ability in MC3T3-E1 cell line of shiitake mushroom extract, and FIG. 22 is a graph showing nutrition for bone calcification formation ability in MC3T3-E1 cell line of vitamin D-shiitake mushroom extract. . As a result of staining after 14 weeks treatment at 0, 10, 50, 100, 200μg/ml concentration, shiitake mushrooms showed 99, 100, 112, 123% bone-calcification ability, and vitamin D-shiitake mushrooms showed 100, 108 , 126, 137% showed the ability to form bone calcification.
추출물 모두 시료를 처리하지 않은 대조군보다 농도가 증가할수록 골석회화 형성능이 증가하였으며 비타민 D-표고버섯이 더 높은 골석회화 형성능 활성을 나타냈다.In both extracts, as the concentration increased compared to the control group that was not treated with the sample, the ability to form bone calcification increased, and vitamin D-shiitake showed higher activity in the formation of bone calcification.
시험예Test example 10: Alkaline 10: Alkaline phosphatasephosphatase (ALP) 활성 측정(ALP) activity measurement
Alkaline phosphatase (ALP)는 거의 모든 조직에 존재하며 특히 골조직에 존재하는 ALP는 골 성장이 활발히 일어날 때 그 활성이 증가한다. 따라서 조골세포 활성을 알아보기 위해 MC3T3-E1 조골세포에서의 ALP 활성에 미치는 영향을 확인하였다. Alkaline phosphatase (ALP) is present in almost all tissues. In particular, ALP, which is present in bone tissue, increases its activity when bone growth occurs actively. Therefore, to investigate the osteoblast activity, the effect on the ALP activity in MC3T3-E1 osteoblasts was confirmed.
조골세포 분화 6일 후 각 농도별 시료를 처리하여 48시간 배양하였다. 배양한 세포는 DPBS로 세척한 후 0.1% Triton X-100을 첨가해 30분간 세포를 lysis 시킨 후 13,000 rpm에서 5분간 원심분리 하였다. 원심분리 후 상층액을 취하고 ALP kit를 사용하여 ALP 활성을 측정하였다. 세포수 차이에 따른 ALP 활성도 변화를 고려하기 위해 총단백질 양은 BSA을 표준 단백질로 BCA protein assay kit를 사용하여 측정하였으며 효소 활성은 대조군에 대한 백분율로 나타냈다.After 6 days of osteoblast differentiation, samples for each concentration were treated and cultured for 48 hours. After washing the cultured cells with DPBS, 0.1% Triton X-100 was added to lyse the cells for 30 minutes, and then centrifuged at 13,000 rpm for 5 minutes. After centrifugation, the supernatant was taken and ALP activity was measured using an ALP kit. In order to consider the change in ALP activity according to the difference in cell number, the total protein amount was measured using BSA as a standard protein using the BCA protein assay kit, and the enzyme activity was expressed as a percentage of the control group.
세포 증식 결과를 토대로 0, 10, 50, 100, 200μg/ml 농도로 7주일간 처리 후 assay kit를 이용하여 활성 측정 및 염색을 통해 활성 변화를 확인하였다. Based on the cell proliferation results, after 7 weeks of treatment at concentrations of 0, 10, 50, 100, 200 μg/ml, activity changes were confirmed through activity measurement and staining using an assay kit.
도 23은 MC3T3-E1 세포주에서 ALP 활성에 관한 표고버섯 추출물의 영향을 나타내는 그라프도이고, 도 24는 MC3T3-E1 세포주에서 ALP 활성에 관한 비타민D-표고버섯 추출물의 영향을 나타내는 그라프도이다. 표고버섯에서는 101, 104, 109, 118% 로 ALP 활성을 나타냈으며, 비타민 D-표고버섯에서는 101, 109, 117, 129%로 ALP 활성을 나타냈다. 23 is a graph showing the effect of shiitake mushroom extract on ALP activity in MC3T3-E1 cell line, and FIG. 24 is a graph showing the effect of vitamin D-shiitake mushroom extract on ALP activity in MC3T3-E1 cell line. Shiitake mushrooms showed ALP activity at 101, 104, 109, and 118%, and vitamin D-shiitake showed ALP activity at 101, 109, 117, and 129%.
추출물 모두 시료를 처리하지 않은 대조군보다 농도가 증가할수록 ALP 활성이 증가하였으며 비타민 D-표고버섯이 더 높은 활성을 나타냈다.In both extracts, the ALP activity increased as the concentration increased, and vitamin D-shiitake showed higher activity than that of the control group that was not treated with the sample.
시험예Test example 11: Collagen 합성에 미치는 영향 분석 11: Analysis of effect on Collagen synthesis
Collagen은 인체 각 결합 조직에 분포하는 중요한 단백질로서, 조골세포에 의해 주로 합성되고 전체 골단백의 85∼90% 를 차지한다. 추출물이 조골세포의 분화를 증가시키는지 알아보기 위해 조골세포의 collagen 합성능을 측정하였다.Collagen is an important protein distributed in each connective tissue of the human body. It is mainly synthesized by osteoblasts and accounts for 85-90% of the total bone protein. To determine whether the extract increases the differentiation of osteoblasts, the ability of osteoblasts to synthesize collagen was measured.
조골세포의 collagen 합성능력은 Tullberg-Reinert와 Jundt의 방법으로 측정하였다[참조문헌: Tullberg-Reinert H, Jundt G. 1999. In situ measurement of collagen synthesis by human bone cells with a sirius red-based colorimetric microassay: effects of transforming growth factor beta2 and ascorbic acid 2-phosphate. Histochem Cell Biol 112: 271-276]. The collagen synthesis ability of osteoblasts was measured by the method of Tullberg-Reinert and Jundt [Reference: Tullberg-Reinert H, Jundt G. 1999. In situ measurement of collagen synthesis by human bone cells with a sirius red-based colorimetric microassay: effects of transforming growth factor beta2 and ascorbic acid 2-phosphate. Histochem Cell Biol 112: 271-276].
조골세포 분화 6일 후 각 농도별 시료를 처리하여 48시간 배양하였다. 배양한 세포는 DPBS로 세척한 후 주로 동물조직 고정에 사용되는 bouin's fluid (saturated picric acid : 35% formaldehyde : glacial acetic acid＝15:5:1)로 1시간 고정시켰다. 고정 후 3차 증류수로 세척한 후 건조하여 sirius red 염색액(1 mg/mL saturated picric acid)으로 1시간 동안 흔들어주며 염색하고 0.01 NHCl로 세척 후 0.1 N NaOH 0.2 mL을 첨가하여 용해시켜 ELISA reader를 사용하여 550 nm에서 흡광도를 측정하였다. Collagen 함량(μg per 2 x 104 cell)은 대조군에 대한 백분율로 나타냈다.After 6 days of osteoblast differentiation, samples for each concentration were treated and cultured for 48 hours. Cultured cells were washed with DPBS and fixed for 1 hour with bouin's fluid (saturated picric acid: 35% formaldehyde: glacial acetic acid = 15:5:1), which is mainly used for fixing animal tissues. After fixation, wash with 3rd distilled water, dry, shake with sirius red staining solution (1 mg/mL saturated picric acid) for 1 hour, dye, wash with 0.01 NHCl, add 0.1 N NaOH, 0.2 mL, and dissolve. The absorbance was measured at 550 nm. Collagen content (μg per 2 x 10 4 cells) was expressed as a percentage of the control group.
도 25는 MC3T3-E1 세포주에서 콜라겐 합성에 미치는 추출물의 영향을 나타낸 그라프도이고, 도 26은 MC3T3-E1 세포주에서 콜라겐 합성에 미치는 비타민D-추출물의 영향을 나타낸 그라프도이다. 0, 10, 50, 100, 200μg/ml 농도로 7주일간 처리 후 염색하여 확인한 결과, 표고버섯에서는 105, 110, 121, 135% 로 콜라겐 합성능을 나타냈으며, 비타민 D-표고버섯에서는 104, 117, 128, 149%로 콜라겐 합성능을 나타냈다. 25 is a graph showing the effect of the extract on collagen synthesis in the MC3T3-E1 cell line, and FIG. 26 is a graph showing the effect of the vitamin D-extract on collagen synthesis in the MC3T3-E1 cell line. As a result of staining after 7 weeks treatment at 0, 10, 50, 100, 200μg/ml concentration, shiitake mushrooms showed collagen synthesis ability of 105, 110, 121, 135%, vitamin D-shiitake mushrooms 104, 117 , 128, 149% showed collagen synthesis ability.
추출물 모두 시료를 처리하지 않은 대조군보다 농도가 증가할수록 콜라겐 합성능이 증가하였으며 비타민 D-표고버섯이 더 높은 콜라겐 합성능 활성을 나타냈다.In both extracts, as the concentration increased, the collagen synthesis ability increased as the concentration increased, and vitamin D-shiitake showed higher collagen synthesis activity.
시험예Test example 12: 12: osteocalcinosteocalcin 생성에 미치는 영향 분석 Analysis of impact on production
osteocalcin은 비타민 K 의존성 단백질로써 조골세포에서 합성된다. osteocalcin은 완전 분화된 osteoblast에서 발현되며 분화단계 중 가장 늦게 발현되는 biomarker로 골형성 여부를 확인 할 수 있는 요소이기도 하다.Osteocalcin is a vitamin K-dependent protein synthesized in osteoblasts. Osteocalcin is expressed in fully differentiated osteoblasts and is the latest biomarker during the differentiation stage and is a factor that can confirm bone formation.
Osteocalcin과 BMP-2 은 LISA kit를 활용하여 함량을 측정하였다. 다양한 농도에서 샘플을 처리한 세포를 배양한 배지를 수거하여 monoclonal osteocalcin 및 BMP-2 antibody와 반응시킨 후 horseradish peroxidase conjugated streptavidin 를 처리하여 horseradish peroxidase substrate(tetramethylbenzidine)을 450 nm에서 흡광도를 측정하였다.The contents of osteocalcin and BMP-2 were measured using the LISA kit. The culture medium of the cells treated with samples at various concentrations was collected, reacted with monoclonal osteocalcin and BMP-2 antibody, and then treated with horseradish peroxidase conjugated streptavidin to measure the absorbance of horseradish peroxidase substrate (tetramethylbenzidine) at 450 nm.
도 27은 표고버섯 추출물의 MC3T3-E1 세포주에서 오스테오칼신 분비에 관한 영향을 나타내는 그라프도이고, 도 28은 비타민D-표고버섯 추출물의 MC3T3-E1 세포주에서 오스테오칼신 분비에 관한 영향을 나타내는 그라프도이다. 0, 10, 50, 100, 200μg/ml 농도로 7주일간 처리 후확인한 결과, 표고버섯에서는 109, 120, 137, 161% 로 osteocalcin 생성이 증가하였으며, 비타민 D-표고버섯에서는 118, 129, 150, 174%로 osteocalcin 생성이 증가하였다. 추출물 모두 시료를 처리하지 않은 대조군보다 농도가 증가할수록 osteocalcin 생성이 증가하였으며 비타민 D-표고버섯이 osteocalcin 생성이 더 높음을 확인하였다.Figure 27 is a graph showing the effect on the secretion of osteocalcin in the MC3T3-E1 cell line of shiitake mushroom extract, and Figure 28 is a graph showing the effect on the secretion of osteocalcin in the MC3T3-E1 cell line of vitamin D-shiitake mushroom extract. As a result of checking after 7 weeks treatment at 0, 10, 50, 100, 200μg/ml concentration, osteocalcin production increased to 109, 120, 137, 161% in shiitake mushrooms, and 118, 129, 150, in vitamin D-shiitake mushrooms. Osteocalcin production increased by 174%. It was confirmed that osteocalcin production increased as the concentration increased in both extracts than in the control group that was not treated with the sample.
시험예Test example 13: 13: 관련인자Related factors 발현 시험 Expression test
Preosteoblast에서 mature osteoblast로 분화하는 과정에서 초기 분화 인자인 ALP, 조골세포 분화 조절인자인 RUNX2와 typeⅠcollagen등의 물질을 합성하여 골 형성을 촉진하며, mature osteoblast에서 골세포화 되는 과정에서 BMP (Bone morphogenetic proteins), OCN (osteocalcin)등 다양한 인자들을 발현시켜 골세포의 석회화를 유도하는 것으로 알려져 있다.In the process of differentiation from preosteoblast to mature osteoblast, bone formation is promoted by synthesizing substances such as ALP, an early differentiation factor, RUNX2, and type I collagen, an osteoblast differentiation regulator, and BMP (Bone morphogenetic proteins ), OCN (osteocalcin), and other factors are known to induce calcification of bone cells.
시료 처리 후 배양된 세포는 lysis buffer (50 mM tris-Cl, 150 mM NaCl, 5mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1% deoxycholate, protease inhibitors)를 이용하여 용해하고 원심분리하여 순수한 단백질을 얻었다. 단백질은 BCA Protein assay kit를 사용하여 정량하고 동량의 단백질은 10% SDS-polyacrylamide gel에서 분리한다. 분리된 단백질은 PVDF membrane으로 옮기고 PVDF membrane은 5% non-fat drymilk를 처리하여 비특이 단백질이 붙는 것을 방지하였다. 그리고 1차 항체 및 2차 항체를 처리한 후 TBS-T 완충용액으로 PVDF막을 세척하여 enhanced chemiluminescence를 이용해 단백질 발현을 관찰하였다. Cells cultured after sample treatment were prepared using lysis buffer (50 mM tris-Cl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1% deoxycholate, protease inhibitors). It was dissolved and centrifuged to obtain pure protein. Protein is quantified using the BCA Protein Assay Kit, and the same amount of protein is isolated on a 10% SDS-polyacrylamide gel. The separated protein was transferred to the PVDF membrane and the PVDF membrane was treated with 5% non-fat drymilk to prevent non-specific proteins from sticking to each other. In addition, after treatment with the primary and secondary antibodies, the PVDF membrane was washed with a TBS-T buffer solution, and protein expression was observed using enhanced chemiluminescence.
조골세포에 미치는 영향에 대하여 ALP 활성, 골석회화능을 측정한 결과를 토대로 50, 100, 200μg/ml 처리한 다음 단백질 발현 변화를 western blot을 이용하여 측정하였다. Based on the results of measuring ALP activity and bone calcification ability for the effect on osteoblasts, 50, 100, 200 μg/ml treatment was performed, and then protein expression changes were measured using western blot.
도 29는 MC3T3-E1 세포주에서 오스테오칼신 분비에 관한 비타민D-추출물의 영향을 나타낸 사진이다. RUNX2와 typeⅠ collagen 발현의 경우 대조군 대비 추출물 농도가 증가할수록 단백질 발현이 증가하는 경향을 보였다. 또한 골세포화 되는 과정에 중요하게 발현되는 BMP2 측정한 결과, 대조군 대비 추출물 농도가 증가할수록 단백질 발현이 증가하는 경향을 보였다. 비타민 D-표고버섯 추출물이 표고버섯 추출물보다 조골세포에 초기분화에 발현하는 typeⅠcollagen , RUNX 2 단백질 발현을 증가시켰으며 성숙 조골세포에서 발현되는 BMP2 또한 단백질 발현을 촉진시켰다. 29 is a photograph showing the effect of vitamin D-extract on the secretion of osteocalcin in MC3T3-E1 cell line. In the case of RUNX2 and type I collagen expression, protein expression tended to increase as the extract concentration increased compared to the control group. In addition, as a result of measuring BMP2, which is important in the process of osteogenesis, protein expression tended to increase as the extract concentration increased compared to the control group. Vitamin D-shiitake extract increased the expression of typeⅠcollagen and
이상과 같은 본 연구의 결과는 표고버섯의 골 대사 개선 기능성에 대한 과학적 입증자료로 활용될 수 있으며, 이러한 기능성 정보는 향후 표고버섯의 산업적 활용을 위한 근거자료가 되고 궁극적으로 관련 시장의 수요확대를 통해 농가 소득 증진에 기여. 표고버섯 추출물의 유효성분을 규명하여 유효성분 고함량 제조를 통해 골 질환 예방에 효과적인 기능성 식품 개발에 대한 기대가 크고, 표고버섯의 특화된 골 대사 개선 기능성을 분석하여 골 질환뿐만 아니라 다양한 분야로의 응용이 확대될 것으로 기대된다.The results of this study as described above can be used as scientific proof data for the function of improving bone metabolism of shiitake mushrooms, and this functional information will serve as the basis for industrial use of shiitake mushrooms in the future and ultimately increase demand in related markets. Contribute to the increase of farm household income. Expectations are high for the development of functional foods that are effective for preventing bone diseases by identifying the active ingredients of shiitake mushroom extract, and by analyzing the specialized function of improving bone metabolism of shiitake mushrooms, it is applied to various fields as well as bone diseases. Is expected to expand.
또한, 표고버섯을 소재로 한 연구개발은 국내에서 생산되는 농산물을 이용한 건강식품시장의 활성화에 밑거름이 될 것이며, 장흥표고의 활용성 증대 및 제품 다양화에 기여할 것으로 기대된다.In addition, research and development using shiitake mushrooms will serve as the foundation for the vitalization of the health food market using agricultural products produced in Korea, and is expected to contribute to the increase in the utilization of Jangheung Shiitake and product diversification.
이상, 첨부된 도면을 참조하여 본 발명의 바람직한 일 실시예를 설명하였지만, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면 본 발명의 그 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 일 실시예는 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.In the above, a preferred embodiment of the present invention has been described with reference to the accompanying drawings, but those of ordinary skill in the art to which the present invention pertains can be used in other specific forms without changing the technical spirit or essential features of the present invention. It will be appreciated that it can be implemented. Therefore, it should be understood that the exemplary embodiment described above is illustrative in all respects and is not limiting.
Claims (5)
상기 표고버섯 추출물은 깨끗이 세척하여 건조하여 세절한 다음 원료 중량의 5배 내지 20배의 추출 용매를 더하여 고압멸균솥에 넣고 65 ~ 125℃의 온도에서 2 내지 72시간 동안 가열시켜 추출하여 여과하여 얻어지는 것을 특징으로 하는 골 대사 개선용 조성물.The method according to claim 1,
The shiitake mushroom extract is washed, dried, and minced, and then added to an extraction solvent of 5 to 20 times the weight of the raw material, put in an autoclave, heated at a temperature of 65 to 125°C for 2 to 72 hours, and extracted and filtered. Composition for improving bone metabolism, characterized in that.
상기 추출용매는 물, 탄소수 1 내지 4의 저급 알코올, 다가 알코올 또는 이들의 혼합물로부터 선택된 적어도 어느 하나인 것을 특징으로 하는 골 대사 개선용 조성물.The method according to claim 2,
The extraction solvent is a composition for improving bone metabolism, characterized in that at least one selected from water, a lower alcohol having 1 to 4 carbon atoms, a polyhydric alcohol, or a mixture thereof.
상기 조성물은 식품 또는 의약 조성물인 것을 특징으로 하는 골 대사 개선용 조성물.The method according to claim 1,
The composition is a composition for improving bone metabolism, characterized in that the food or pharmaceutical composition.
상기 조성물은 골다공증 (Osteoporosis), 골연화증 (Osteomalacia), 골감소증 (Osteopenia), 골 위축(Bone atrophy), 섬유성 골이형성증 (Fibrous dysplasia), 파제트병 (Paget's disease), 고칼슘혈증(Hypercalcemia), 뼈의 종양성 파괴 (Neoplastic bone destruction), 및 골 성장장애 (Growth disorder of the bone)로 이루어진 군으로부터 선택되는 하나 이상의 질환을 개선하는 것을 특징으로 하는 골 대사 개선용 조성물.The method according to claim 1,
The composition comprises Osteoporosis, Osteomalacia, Osteopenia, Bone atrophy, Fibrous dysplasia, Paget's disease, Hypercalcemia, and bone A composition for improving bone metabolism, characterized in that it improves at least one disease selected from the group consisting of neoplastic bone destruction, and growth disorder of the bone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020190082873A KR20210006801A (en) | 2019-07-09 | 2019-07-09 | Composition for improving bone metabolism comprising shitake mushroom extracts |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020190082873A KR20210006801A (en) | 2019-07-09 | 2019-07-09 | Composition for improving bone metabolism comprising shitake mushroom extracts |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20210006801A true KR20210006801A (en) | 2021-01-19 |
Family
ID=74237234
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020190082873A KR20210006801A (en) | 2019-07-09 | 2019-07-09 | Composition for improving bone metabolism comprising shitake mushroom extracts |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR20210006801A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118716632A (en) * | 2024-09-04 | 2024-10-01 | 海南华研美健生物科技有限公司 | Polypeptide composition for improving osteoporosis and enhancing bone density and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101359502B1 (en) | 2012-06-18 | 2014-02-10 | 대한민국 | Food composition for prevention and improvement of osteoporosis using silkworm and food comprising the same |
| KR101502465B1 (en) | 2013-10-15 | 2015-03-13 | 한국 한의학 연구원 | A pharmaceutical composition comprising Alpinia Officinarum extracts for prevention and treatment of bone diseases or anti-vascular calcification activity |
-
2019
- 2019-07-09 KR KR1020190082873A patent/KR20210006801A/en not_active Application Discontinuation
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101359502B1 (en) | 2012-06-18 | 2014-02-10 | 대한민국 | Food composition for prevention and improvement of osteoporosis using silkworm and food comprising the same |
| KR101502465B1 (en) | 2013-10-15 | 2015-03-13 | 한국 한의학 연구원 | A pharmaceutical composition comprising Alpinia Officinarum extracts for prevention and treatment of bone diseases or anti-vascular calcification activity |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118716632A (en) * | 2024-09-04 | 2024-10-01 | 海南华研美健生物科技有限公司 | Polypeptide composition for improving osteoporosis and enhancing bone density and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101776071B1 (en) | Composition containing red sword bean extract for anti-aging and whitening | |
| JP4180388B2 (en) | Process for producing biotransformation ginseng composition | |
| WO2009123364A1 (en) | Mineral absorption accelerator and iron deficiency anemia improver or food composition | |
| WO2015190872A1 (en) | Pharmaceutical composition containing spirulina maxima extract as active ingredient for treating and preventing obesity | |
| KR20090077572A (en) | Composition for treating or preventing metabolic syndrome-related diseases comprising desalinized magma seawater | |
| KR101959731B1 (en) | A composition for preventing or treating menopausal disorder comprising extract from young barley leaves | |
| KR20210006801A (en) | Composition for improving bone metabolism comprising shitake mushroom extracts | |
| KR20140060106A (en) | Neuroprotective composition comprising extracts or fractions of seaweed as an active ingredient | |
| KR102372440B1 (en) | Phamaceutical Composition Comprising an Extract of Artemisia scoparia for Preventing or Treating Metabolic Bone Disease-induced Bone Loss | |
| KR102566496B1 (en) | A pharmaceutical composition for preventing or treating osteoporosis comprising MSM, calcium and rosemary extract as an active ingredient | |
| KR101385191B1 (en) | Use of Cichorium intybus extracts for preventing, treating improving muscular atrophy | |
| KR20180020798A (en) | Composition for lowering blood uric acid level comprising Aster glehini extract and vitamin | |
| KR20120018556A (en) | Compositions comprising extracts of sorbus commixta and geranium nepalense for inhibiting osteoclastogenesis and stimulating chondrogenesis | |
| KR20120016960A (en) | Composition comprising laminaria japonica for preventing and treating obesity or hyperlipidemia and atherosclerotic-vascular diseases | |
| KR20200125154A (en) | Composition for improvementing, preventing or treating obesity and metabolic diseases comprising fractions or extract of molokhia leave | |
| US20140199410A1 (en) | Edible brown algae extract with a low iodine content | |
| KR101402929B1 (en) | Pharmaceutical composition and functional food for prevention or treatment of acute renal failure comprising a herbal extract | |
| KR100373928B1 (en) | Plant extract compositions comprising ecdysteroids and iridoids and the use thereof | |
| KR102567725B1 (en) | A composition for bone health comprising fermented-benicasa hispida extract | |
| KR102141426B1 (en) | Pharmacieutical composition comprising Phloretin 4-O-glucoside for prevention and treatment of bone diseases | |
| KR101918206B1 (en) | Composition for preventing or treating osteoporosis comprising compound isolated from Paeonia lactiflora extract as effective component | |
| KR101398737B1 (en) | Composition for preventing and/or treating tumor containing component originating in the bark of tree belonging to the genus acacia | |
| KR102151643B1 (en) | Composition comprising extract of Lentinula edodes for prevention or treatment of bone diseases | |
| KR100494871B1 (en) | Extracts of CHAGA mushroom showing antioxidant and lipid metabolic activity, the extraction method and the use thereof | |
| KR20120016962A (en) | Composition comprising hizikia fusiformis for preventing and treating obesity or hyperlipidemia and atherosclerotic-vascular diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| E601 | Decision to refuse application |