KR101848171B1 - Chinaroot Root Composition For The Prevention And Treatment Of Osteoporosis And Method Of Manufacturing The Same - Google Patents
Chinaroot Root Composition For The Prevention And Treatment Of Osteoporosis And Method Of Manufacturing The Same Download PDFInfo
- Publication number
- KR101848171B1 KR101848171B1 KR1020160156090A KR20160156090A KR101848171B1 KR 101848171 B1 KR101848171 B1 KR 101848171B1 KR 1020160156090 A KR1020160156090 A KR 1020160156090A KR 20160156090 A KR20160156090 A KR 20160156090A KR 101848171 B1 KR101848171 B1 KR 101848171B1
- Authority
- KR
- South Korea
- Prior art keywords
- extract
- osteoporosis
- concentration
- root
- trap
- Prior art date
Links
- 208000001132 Osteoporosis Diseases 0.000 title claims abstract description 54
- 239000000203 mixture Substances 0.000 title claims abstract description 49
- 230000002265 prevention Effects 0.000 title claims description 28
- 238000004519 manufacturing process Methods 0.000 title abstract description 6
- 235000000485 Smilax china Nutrition 0.000 title 1
- 241000533870 Smilax tamnoides Species 0.000 title 1
- 210000002997 osteoclast Anatomy 0.000 claims abstract description 70
- 239000000284 extract Substances 0.000 claims abstract description 55
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000002904 solvent Substances 0.000 claims abstract description 37
- 238000000605 extraction Methods 0.000 claims abstract description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000007864 aqueous solution Substances 0.000 claims abstract description 8
- 102000007591 Tartrate-Resistant Acid Phosphatase Human genes 0.000 claims description 52
- 108010032050 Tartrate-Resistant Acid Phosphatase Proteins 0.000 claims description 52
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 34
- 108010001789 Calcitonin Receptors Proteins 0.000 claims description 30
- 102100038520 Calcitonin receptor Human genes 0.000 claims description 30
- 230000006872 improvement Effects 0.000 claims description 27
- 102000004169 proteins and genes Human genes 0.000 claims description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 21
- 230000004083 survival effect Effects 0.000 claims description 19
- 235000010323 ascorbic acid Nutrition 0.000 claims description 17
- 229960005070 ascorbic acid Drugs 0.000 claims description 17
- 239000011668 ascorbic acid Substances 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- 229930003935 flavonoid Natural products 0.000 claims description 15
- 235000017173 flavonoids Nutrition 0.000 claims description 15
- 150000002215 flavonoids Chemical class 0.000 claims description 15
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 claims description 14
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 13
- 230000002829 reductive effect Effects 0.000 claims description 11
- 229940074391 gallic acid Drugs 0.000 claims description 7
- 235000004515 gallic acid Nutrition 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims 1
- 238000010298 pulverizing process Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 44
- 230000005764 inhibitory process Effects 0.000 abstract description 7
- 241000605422 Asparagus asparagoides Species 0.000 abstract 2
- 210000000988 bone and bone Anatomy 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 19
- 230000004069 differentiation Effects 0.000 description 16
- 102000014128 RANK Ligand Human genes 0.000 description 15
- 108010025832 RANK Ligand Proteins 0.000 description 15
- 208000006386 Bone Resorption Diseases 0.000 description 12
- 230000024279 bone resorption Effects 0.000 description 12
- 239000003814 drug Substances 0.000 description 7
- 230000011164 ossification Effects 0.000 description 7
- 210000000963 osteoblast Anatomy 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 230000004952 protein activity Effects 0.000 description 7
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 6
- 230000003078 antioxidant effect Effects 0.000 description 6
- 239000011575 calcium Substances 0.000 description 6
- 229910052791 calcium Inorganic materials 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 238000005259 measurement Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 102000008108 Osteoprotegerin Human genes 0.000 description 4
- 108010035042 Osteoprotegerin Proteins 0.000 description 4
- 239000003963 antioxidant agent Substances 0.000 description 4
- 235000006708 antioxidants Nutrition 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- XXUPLYBCNPLTIW-UHFFFAOYSA-N octadec-7-ynoic acid Chemical compound CCCCCCCCCCC#CCCCCCC(O)=O XXUPLYBCNPLTIW-UHFFFAOYSA-N 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 150000008442 polyphenolic compounds Chemical class 0.000 description 4
- 235000013824 polyphenols Nutrition 0.000 description 4
- 229960005322 streptomycin Drugs 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- 244000184734 Pyrus japonica Species 0.000 description 3
- 102000010498 Receptor Activator of Nuclear Factor-kappa B Human genes 0.000 description 3
- 108010038036 Receptor Activator of Nuclear Factor-kappa B Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 210000002805 bone matrix Anatomy 0.000 description 3
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 description 3
- 235000005487 catechin Nutrition 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 229950001002 cianidanol Drugs 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 2
- 229940122361 Bisphosphonate Drugs 0.000 description 2
- 208000020084 Bone disease Diseases 0.000 description 2
- 206010065687 Bone loss Diseases 0.000 description 2
- 229940078581 Bone resorption inhibitor Drugs 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 206010017076 Fracture Diseases 0.000 description 2
- 231100000002 MTT assay Toxicity 0.000 description 2
- 238000000134 MTT assay Methods 0.000 description 2
- 102000003945 NF-kappa B Human genes 0.000 description 2
- 108010057466 NF-kappa B Proteins 0.000 description 2
- 102100034404 Nuclear factor of activated T-cells, cytoplasmic 1 Human genes 0.000 description 2
- 101710151542 Nuclear factor of activated T-cells, cytoplasmic 1 Proteins 0.000 description 2
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 2
- 229930003316 Vitamin D Natural products 0.000 description 2
- 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 2
- 238000002835 absorbance Methods 0.000 description 2
- 150000004663 bisphosphonates Chemical class 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 230000004097 bone metabolism Effects 0.000 description 2
- 229940037448 calcitonin preparations Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000001378 electrochemiluminescence detection Methods 0.000 description 2
- 238000002481 ethanol extraction Methods 0.000 description 2
- 239000000469 ethanolic extract Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 230000003054 hormonal effect Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- IYRMWMYZSQPJKC-UHFFFAOYSA-N kaempferol Chemical compound C1=CC(O)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 IYRMWMYZSQPJKC-UHFFFAOYSA-N 0.000 description 2
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 210000005087 mononuclear cell Anatomy 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 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 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 235000019166 vitamin D Nutrition 0.000 description 2
- 239000011710 vitamin D Substances 0.000 description 2
- 150000003710 vitamin D derivatives Chemical class 0.000 description 2
- 229940046008 vitamin d Drugs 0.000 description 2
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- OGSPWJRAVKPPFI-UHFFFAOYSA-N Alendronic Acid Chemical compound NCCCC(O)(P(O)(O)=O)P(O)(O)=O OGSPWJRAVKPPFI-UHFFFAOYSA-N 0.000 description 1
- 208000010392 Bone Fractures Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102000004171 Cathepsin K Human genes 0.000 description 1
- 108090000625 Cathepsin K Proteins 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- UBSCDKPKWHYZNX-UHFFFAOYSA-N Demethoxycapillarisin Natural products C1=CC(O)=CC=C1OC1=CC(=O)C2=C(O)C=C(O)C=C2O1 UBSCDKPKWHYZNX-UHFFFAOYSA-N 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- DBVJJBKOTRCVKF-UHFFFAOYSA-N Etidronic acid Chemical compound OP(=O)(O)C(O)(C)P(O)(O)=O DBVJJBKOTRCVKF-UHFFFAOYSA-N 0.000 description 1
- 208000008924 Femoral Fractures Diseases 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 201000002980 Hyperparathyroidism Diseases 0.000 description 1
- 208000011327 Increased bone mineral density Diseases 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 206010030216 Oesophagitis Diseases 0.000 description 1
- 206010030247 Oestrogen deficiency Diseases 0.000 description 1
- 102000004067 Osteocalcin Human genes 0.000 description 1
- 108090000573 Osteocalcin Proteins 0.000 description 1
- 102000009890 Osteonectin Human genes 0.000 description 1
- 108010077077 Osteonectin Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 108010071563 Proto-Oncogene Proteins c-fos Proteins 0.000 description 1
- 102000007568 Proto-Oncogene Proteins c-fos Human genes 0.000 description 1
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 description 1
- 244000178231 Rosmarinus officinalis Species 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- ABUGVBRDFWGJRD-CHOYNLESSA-N [9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-methyloxolan-2-yl]-2-(2,4-dinitrophenyl)sulfanylpurin-6-yl] [hydroxy(phosphonooxy)phosphoryl] hydrogen phosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](C)O[C@H]1N1C2=NC(SC=3C(=CC(=CC=3)[N+]([O-])=O)[N+]([O-])=O)=NC(OP(O)(=O)OP(O)(=O)OP(O)(O)=O)=C2N=C1 ABUGVBRDFWGJRD-CHOYNLESSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 229940062527 alendronate Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 230000037182 bone density Effects 0.000 description 1
- 239000002617 bone density conservation agent Substances 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 230000034127 bone morphogenesis Effects 0.000 description 1
- 230000010072 bone remodeling Effects 0.000 description 1
- 230000008416 bone turnover Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 230000003913 calcium metabolism Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 235000018823 dietary intake Nutrition 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 208000006881 esophagitis Diseases 0.000 description 1
- 229940009626 etidronate Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 208000001130 gallstones Diseases 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 235000008777 kaempferol Nutrition 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000004571 lime Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 208000018773 low birth weight Diseases 0.000 description 1
- 231100000533 low birth weight Toxicity 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000009245 menopause Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- UXOUKMQIEVGVLY-UHFFFAOYSA-N morin Natural products OC1=CC(O)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UXOUKMQIEVGVLY-UHFFFAOYSA-N 0.000 description 1
- 210000001721 multinucleated osteoclast Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000002346 musculoskeletal system 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
- 230000030991 negative regulation of bone resorption Effects 0.000 description 1
- 230000020395 negative regulation of osteoclast differentiation Effects 0.000 description 1
- 238000009806 oophorectomy Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000000879 optical micrograph Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001599 osteoclastic effect Effects 0.000 description 1
- 210000004409 osteocyte Anatomy 0.000 description 1
- 230000000010 osteolytic effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 231100000208 phytotoxic Toxicity 0.000 description 1
- 230000000885 phytotoxic effect Effects 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000018398 positive regulation of bone resorption Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 229960001285 quercetin Drugs 0.000 description 1
- 235000005875 quercetin Nutrition 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- GZUITABIAKMVPG-UHFFFAOYSA-N raloxifene Chemical compound C1=CC(O)=CC=C1C1=C(C(=O)C=2C=CC(OCCN3CCCCC3)=CC=2)C2=CC=C(O)C=C2S1 GZUITABIAKMVPG-UHFFFAOYSA-N 0.000 description 1
- 229960004622 raloxifene Drugs 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 229960000819 sodium nitrite Drugs 0.000 description 1
- 206010041569 spinal fracture Diseases 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 229960004319 trichloroacetic acid Drugs 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/90—Smilacaceae (Catbrier family), e.g. greenbrier or sarsaparilla
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/05—Phenols
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Mycology (AREA)
- Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
본 발명은 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물 및 이의 제조방법에 관한 것으로, 보다 상세하게는 부작용을 최소화시키면서 파골세포 활성의 억제를 통하여 골다공증 예방 및/또는 개선 효과를 극대화 시킬 수 있는 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물 및 이의 제조방법에 관한 것이다.More particularly, the present invention relates to a composition for preventing and / or improving osteoporosis, which is capable of minimizing side effects and preventing osteoporosis and / And a method for producing the same.
일반적으로 골(bone)은 근육과 연합되어 있는 석회화된 결체조직으로서 표면은 두껍고 단단한 석회화 조직으로 신체균형을 이루는 지주역할을 하며 장기를 보호한다.In general, the bone is a calcified connective tissue associated with the muscles. The surface is thick and hard calcified tissue, which serves as a body balance and protects the organs.
골의 안쪽은 골수조직으로 칼슘대사의 중심이 되는 부위이다. 골은 콜라겐(collagen), 오스테오칼신(osteocalcin), 오스테오넥틴(osteonectin) 등 유기질(organic substance)과 칼슘, 인, 불소 등의 무기질(inorganic substance) 및 수분으로 구성되어 있다.The inside of bone is bone marrow tissue, which is the center of calcium metabolism. The bone consists of an organic substance such as collagen, osteocalcin, osteonectin, and inorganic substance such as calcium, phosphorus, and fluorine, and moisture.
생체 내 골 대사는 골 형성(bone formation)을 담당하는 조골세포와 골 흡수(bone resorption)를 담당하는 파골세포의 작용으로 이루어져 있으며, 이들 세포의 기능이 균형을 이루어 향상성을 유지한다.In vivo bone metabolism is composed of osteoblasts responsible for bone formation and osteoclasts responsible for bone resorption. These cells function in balance to maintain their improvement.
따라서 골 대사에서는 이들 사이의 균형이 중요하다. 골 조직은 지속적으로 재형성되는 활발한 대사조직으로 노화된 뼈를 파골세포에 의해 용해하는데, 뼈의 표면에 부착하여 산화 분해효소를 분비함으로써 뼈를 구성하는 뼈 기질(matrix)를 제거한다.Therefore, the balance between bone metabolism is important. Bone tissue is an active metabolic tissue that is constantly reformed. It dissolves the aged bone by osteoclast. It attaches to the surface of bone and secretes oxidase, which removes the bone matrix constituting bone.
반면에 새로운 뼈의 형성은 조골세포(osteoblast)가 칼슘과 인을 분비하여 골격을 형성한다. 생체 내에서 이들 세포의 항상성 유지에 의해 일어나 골 흡수와 골 형성은 근골격계의 정상적인 구조를 유지하는 중요하다.On the other hand, the formation of new bone is caused by osteoblasts secreting calcium and phosphorus to form a skeleton. It is important to maintain the normal structure of the musculoskeletal system by osteosynthesis and osteogenesis caused by the homeostasis of these cells in vivo.
골다공증(osteoporosis)은 골흡수와 골형성의 균형이 무너져 발생하는 것으로 골형성보다 과다하게 골흡수가 진행되는데 기인한 질환으로, 골다공증은 골 조직의 석회가 감소 되어 골의 치밀질이 엷어지고 그로 인해 골수강(骨髓腔)이 넓어지고, 증세가 진전됨에 따라 골이 약해지기 때문에 작은 충격에도 골절되기 쉽다. Osteoporosis is a disease caused by the collapse of the balance between bone resorption and bone formation, which is caused by excessive bone resorption over osteogenesis. Osteoporosis is a disease caused by reduced lime of bone tissue and thinness of bone, As the bone marrow cavity is widened and the symptoms progress, the bone is weakened, so it is likely to fracture even in small impacts.
골조직은 조골세포에 의해 형성되고 파골세포에 의해 파괴 흡수가 끊임없이 반복되는 동적인 조직이다. 골의 항상성은 파골 세포(osteoclast)에 의한 골 흡수(bone resoprtion)와 조골세포(osteoblast)에 의한 골 형성(bone formation)의 대등한 작용에 의한 리모델링 과정(bone remodeling)이 지속적으로 조절되어 유지된다.Bone tissue is a dynamic tissue that is formed by osteoblasts and is constantly repetitively absorbed and destroyed by osteoclasts. The homeostasis of bone is maintained by continuously controlling the bone remodeling by the equivalent action of bone resorption by osteoclast and osteoblast formation (bone formation) .
그러나, 파골 세포의 지나친 활성이나 조골세포 활성의 저하는 리모델링 과정의 불균형을 초래하여, 골다공증(osteoporosis)과 같은 성인 골격계 질환을 유도한다.However, excessive activity of osteoclasts or a decrease in osteoblast activity leads to an imbalance in the remodeling process, leading to adult skeletal diseases such as osteoporosis.
이러한 불균형을 해소하기 위해서 일반적으로 파골 세포의 지나친 활성을 억제하거나, 조골세포의 활성을 촉진시키거나, 또는 파골 세포의 활성을 억제하고 조골세포의 활성을 촉진하는 방법이 사용되고 있으며, 이들은 골다공증의 치료에 대한 효과적인 치료적 접근 방법으로 인지되고 있다.In order to alleviate this imbalance, methods for suppressing excessive activity of osteoclasts, promoting osteoclast activity, inhibiting osteoclast activity and promoting osteoclast activity are generally used, and they are used for treatment of osteoporosis As well as for the treatment of the disease.
파골세포는 조혈모세포의 단핵구로부터 유래되는 세포로서, 마우스의 RAW264.7 단핵구 세포는 RANKL(receptor activator of nuclear factor κB (RANK) ligand)에 의해 융합하여 다핵 파골세포(multinucleated osteoclasts)로 분화된다.(참조: Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003 May 15;423(6937):337-42).Bone marrow cells are derived from mononuclear cells of hematopoietic stem cells. Mouse RAW264.7 mononuclear cells are differentiated into multinucleated osteoclasts by fusion with RANKL (receptor activator of nuclear factor κB (RANK) ligand). See: Boyle WJ, Simonet WS, Lacey DL, Osteoclast differentiation and activation, Nature, 2003 May 15; 423 (6937): 337-42).
파골세포를 활성화시키는 중요한 씨토카인은 Receptor activator of nuclear fractor-κB ligand (RANKL)로서 조골세포 또는 활성화된 면역세포에서 생성된다.The important cytokine that activates osteoclasts is the Receptor activator of nuclear factor-κB ligand (RANKL), which is produced in osteoblasts or in activated immune cells.
생성된 RANKL은 파골세포 및 전구세포에 위치한 수용체(RANK)와 결합하여 파골세포의 형성과 활성을 촉진시킨다.The resulting RANKL binds to a receptor (RANK) located in osteoclast and progenitor cells to promote osteoclast formation and activity.
또한 RANKL에 대한 생체 내 길항제인 Osteoprotegerin (OPG)가 존재하고 있다. 결국, RANKL과 OPG의 균형에 의하여 파골세포의 형성과 활성이 조절되는 것이다.There is also an in vivo antagonist, Osteoprotegerin (OPG), for RANKL. Finally, osteoclast formation and activity are regulated by the balance of RANKL and OPG.
그러나, 골 흡수를 자극하는 원인(에스트로겐 결핍, 부갑상선 호르몬 항진증 등)이 발생하게 되면 OPG와 RANKL의 균형이 깨지게 되어 파골세포가 활성화되어 골 흡수가 촉진되는 것이다. 활성화된 파골세포는 골 표면에 부착하여 강한 골 용해물질을 분비한다. 골 단백 용해제 중 하나인 카텝신 (cathepsin) K가 파골세포 특이적으로 생성 분비되며, 풍부한 칼시토닌 수용체(calcitonin receptor, Cal-R)와 TRAP이 존재하며 실제적으로 골 흡수 작용을 하는 산 생성이 활발하고, acting ring을 형성하여 골 기질을 흡수한다고 알려졌다.However, when the cause of stimulation of bone resorption (estrogen deficiency, hyperparathyroidism, etc.) occurs, balance of OPG and RANKL is broken and osteoclast is activated and bone resorption is promoted. Activated osteoclasts attach to the bone surface and secrete strong osteolytic substances. One of the bone protein solubilizers, cathepsin K, is produced and secreted specifically by osteoclasts. There are abundant calcitonin receptors (Cal-R) and TRAP. , and an activating ring to absorb bone matrix.
따라서 이들의 억제를 통하여 골 흡수 억제 효과를 이끌어낼 수 있을 것이다. 골 흡수를 억제하기 위해 파골 전구세포에서 파골세포로의 분화를 차단하는 분화 과정의 초기단계 저해와 분화된 파골세포의 사멸을 유도하여 골 흡수 과정을 저해하는 연구가 진행되고 있다.Therefore, the inhibition of bone resorption may be achieved through their inhibition. In order to inhibit bone resorption, studies are under way to inhibit the bone resorption process by inducing early stage inhibition of differentiation blocking osteoclast differentiation from osteoclast precursor cells and differentiating osteoclast cell death.
골다공증은 그 증세 자체보다는 골의 약화에 따라 용이하게 초래되는 각종 골절, 특히 대퇴골 골절 또는 척추골절 등이 장기간 활동을 제한하여 건강한 생활을 영위할 수 없고, 결과적으로 노인층 사망의 15%에 대한 원인제공을 하는 것으로 알려져 있다.Osteoporosis is a cause of 15% of the deaths of the elderly as a result of various fractures, especially femoral fractures or vertebral fractures, which are easily caused by the weakening of the bone rather than the symptom itself, .
골량은 유전적 요인, 영양 섭취, 호르몬의 변화, 운동 및 생활 습관의 차이 등 여러 가지 요인들에 의해 영향을 받으며, 골다공증의 원인으로는 노령, 운동 부족, 저체중, 흡연, 저칼슘 식이, 폐경, 난소 절제 등이 알려져 있다.The bone mass is affected by various factors such as genetic factors, nutritional intake, hormonal changes, exercise and lifestyle differences, and the causes of osteoporosis include age, lack of exercise, low birth weight, smoking, low calcium diet, Ovariectomy and the like are known.
한편 개인 차이는 있지만 백인보다는 흑인이 골 재흡수 수준(bone resorption level)이 낮아 골량이 더 높으며, 대개 골량은 14 ~ 18세에 가장 높고 노후에는 1 년에 약 1 %씩 감소한다. 특히 여성의 경우 30 세 이후부터 골 감소가 지속적으로 진행되며, 폐경기에 이르면 호르몬 변화에 의해 골 감소가 급격히 진행된다.Although there are individual differences, blacks have lower bone resorption levels than blacks, and bone mass is higher. In general, bone mass is highest at 14-18 years old and decreases about 1% per year at old age. Especially in women, bone reduction continues after 30 years of age, and bone turnover is rapidly progressed by hormonal changes in menopause.
이와 같이 골다공증은 정도에 차이는 있으나 노년층, 특히 폐경기 이후의 여성에게 있어서는 피할 수 없는 증상으로, 선진국에서는 인구가 노령화됨에 따라 골다공증 및 그 치료제에 대한 관심이 점차 증가되고 있다.Thus, osteoporosis is an unavoidable symptom for elderly people, especially postmenopausal women. In the developed countries, as population ages, interest in osteoporosis and its therapeutic agents is gradually increasing.
또한, 전세계적으로 골질환 치료와 관련되어 약 1,300억 달러의 시장이 형성되어 있는 것으로 알려져 있으며 앞으로 더 증가할 것으로 예상되기 때문에, 세계적인 각 연구 기관과 제약회사에서는 골질환 치료제 개발에 많은 투자를 하고 있고 현재 골흡수 억제제의 개발이 활발히 진행되고 있다.It is also known that there is a market of about $ 130 billion related to the treatment of bone diseases worldwide and it is expected to increase further in the future. Therefore, each research institute and pharmaceutical company in the world invests heavily in the development of bone disease drugs Currently, the development of bone resorption inhibitors is actively underway.
골다공증과 관련하여 과거에는 주로 골의 무기질, 즉 칼슘과 인의 대사이상을 중심으로 그 연구가 진행되어 왔으나, 이의 발병 기전 규명에는 큰 진전을 보지 못하였다.In the past, osteoporosis was mainly related to the bone mineral, calcium and phosphorus metabolism. However, no major progress has been made in the identification of the mechanisms underlying the onset of osteoporosis.
현재 골다공증 치료제로 사용되고 있는 물질로는 비스포스포네이트 제제(알렌드로네이트, 에티드로네이트), 호르몬 제제(랄록시펜), 비타민 D 제제, 칼시토닌 제제, 칼슘 제제 등이 있다.Currently, there are bisphosphonates (alendronate, etidronate), hormone preparations (raloxifene), vitamin D preparations, calcitonin preparations and calcium preparations.
그러나, 비스포스포네이트 제제는 흡수율이 떨어지며 복용방법이 까다롭고 식도염을 유발시키며, 호르몬 제제는 평생 복용하여야 하며 장기 투여할 경우 유방암, 자궁암, 담석 및 혈전증 등의 부작용이 나타나고, 비타민 D 제제는 고가이며 효과가 확실하지 않고, 칼시토닌 제제는 고가이며 투여방법이 어렵고, 칼슘제제는 부작용은 적지만 치료보다는 예방효과에 국한되는 단점이 있다.However, bisphosphonate preparations have poor water uptake and are difficult to take and cause esophagitis, and hormone preparations should be used for a lifetime. When administered for a long time, adverse effects such as breast cancer, uterine cancer, gallstone and thrombosis appear. Vitamin D preparations are expensive and effective Calcitonin preparations are expensive, difficult to administer, and calcium preparations have few side effects, but they are limited to preventive effects rather than treatment.
골다공증은 약물의 단기 투여만으로는 치료할 수 없으며 약물의 장기 투여가 필수적이다. 따라서, 약물을 장기투여할 때에 상기와 같은 부작용이 없고 우수한 약효를 갖는 새로운 물질의 필요성이 요구되고 있다.Osteoporosis can not be treated by only short-term administration of the drug, and long-term administration of the drug is essential. Therefore, there is a need for a new substance having excellent drug efficacy without the above-mentioned side effects when the drug is administered for a long period of time.
근래에는 이러한 부작용을 최소하기 위하여 독성이 적고, 일상생활에서 식용으로 섭취가능한 식품과 약용식물 등 천연물 소재를 이용하여 골 손실의 최소화와 더불어 골 형성을 촉진시킬 수 있는 골다공증 예방 또는 치료 물질 개발에 대한 응용 연구가 다양하게 시도되고 있다.Recently, in order to minimize such side effects, there has been proposed a method for preventing or treating osteoporosis, which can minimize bone loss and promote bone formation by using natural materials such as foods and medicinal plants that are low in toxicity and can be consumed for food in daily life Application studies have been variously tried.
골 흡수 저하 관련 약용식물의 연구로써 두충, 노각나무, 삼백초, 작약 등이 RANKL로 유도된 파골세포 분화를 억제시켜 골흡수가 저하된다고 하였다(Kwak et al., 2008, Tsai et al., 2008). (Kwak et al., 2008, Tsai et al., 2008). In this study, we investigated the effects of osteoclast-induced osteoclast differentiation on osteoclast- .
최근, Quercetin과 Kaempferol의 파골세포의 골 흡수 억제 효과나 대두를 이용한 조골세포 골 형성 활성 검색, 두충의 골다공증 효과에 대한 연구가 활발하게 진행되고 있으며, 산화스트레스 증가와 골밀도 간 유의한 음의 상관 관계가 있는 것으로 보고된 바(Basu et al., 2001), 산화스트레스가 파골세포에 직접적으로 작용하여 분화와 활성을 증가시킴으로써 골 흡수가 촉진된다고 알려졌고(Yang et al., 1998), 항산화능을 갖는 폴리페놀이나 플라보노이드가 많이 함유된 천연물 유래 약용 식물, 인삼의 사포닌과 같은 특정 성분이 파골세포 분화 및 기능을 억제하여 골흡수 억제제로서 기능을 하는 것으로 보고되었다. 특히, 골다공증 여성의 경우 혈중 항산화제의 농도가 감소되고, 항산화 비타민 섭취로 인하여 골밀도를 증가시켰다고 보고하였다. Recently, studies on the osteoclastic bone resorption inhibition effect of quercetin and kaempferol, osteoblast osteogenesis activity using soybean, and osteoporosis effect have been actively carried out, and a negative correlation between increase in oxidative stress and bone mineral density (Yang et al., 1998), it has been reported that oxidative stress acts directly on osteoclasts and promotes bone resorption by increasing differentiation and activity (Yang et al., 1998) It has been reported that certain components such as polyphenol and natural plant-derived medicinal plants containing large amount of flavonoids and saponin of ginseng inhibit osteoclast differentiation and function and function as a bone resorption inhibitor. In particular, women with osteoporosis reported decreased levels of antioxidants in the blood and increased bone mineral density due to antioxidant vitamin intake.
따라서, 항산화 성분을 가진 유효 약용식물의 안전한 천연물 소재를 이용하여 골 손실의 최소화시켜 골밀도 증가를 통한 골다공증을 예방할 수 있는 천연 치료 물질 발굴이 절실하게 요구된다.Therefore, it is urgently required to find natural therapeutic substances that can prevent osteoporosis by increasing bone density by minimizing bone loss by using safe natural materials of effective medicinal plants having antioxidant components.
본 발명은 상기와 같은 문제점을 감안하여 안출된 것으로, 본 발명의 제 1목적은, 파골세포의 분화 및 기능저하를 통한 골 흡수 억제 기능을 밝혀냄으로써 골다공증 예방 및 개선제로써 이용 가능한 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물 및 이의 제조방법을 제공하는데 있다.SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and it is a first object of the present invention to provide a method for preventing and improving osteoporosis, which can be used as an agent for preventing and improving osteoporosis, And a method for producing the same.
본 발명의 제 2목적은, 청미래덩굴 뿌리추출물에 페놀, 플라보노이드 및 아스코르브산을 포함시켜 항산화 효과가 우수한 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물 및 이의 제조방법을 제공하는데 있다.It is a second object of the present invention to provide a cheonggyeong vine root composition for prevention and improvement of osteoporosis which contains phenol, flavonoid and ascorbic acid and has excellent antioxidative effect, and a method for producing the same.
상기한 바와 같은 목적을 달성하기 위한 특징에 따르면, 제 1발명은, 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물에 관한 것으로, 물 또는 에탄올수용액 중 어느 하나를 추출 용매로 하여 약학적으로 유효한 청미래덩굴 뿌리추출물로 이루어지되, 상기 청미래덩굴 뿌리추출물은 10 ~ 350㎍/㎖의 농도를 갖고, 상기 뿌리추출물의 농도는 파골세포(osteoclast)의 생존율에 영향을 주지 않으면서, Cal-R(칼시토닌 수용체)의 단백질 및 TRAP(주석산염 저항산성 인산분해효소)의 단백질로 이루어진 군으로부터 선택되는 1종 이상의 발현을 감소시켜 골다공증 예방 및 개선에 사용되는 것을 특징으로 한다.According to an aspect of the present invention for achieving the above object, the first aspect of the present invention relates to a cheonggyeong vine root composition for the prevention and improvement of osteoporosis, wherein a water or ethanol aqueous solution is used as an extraction solvent, (Calcitonin Receptor), without affecting the survival rate of the osteoclast, and the root extract has a concentration of 10 to 350 μg / ml. Protein and a protein of TRAP (tartrate-resistant acid phosphatase), and is used for prevention and improvement of osteoporosis.
삭제delete
삭제delete
제 3발명은, 제 1발명에서, 상기 뿌리추출물 100mg에 대하여, 90 ~ 470mg GAE(gallic acid equivalent)의 페놀과, 1,100 ~ 1,800mg의 플라보노이드와, 18 ~ 35mg의 아스코르브산이 더 포함되는 것을 특징으로 한다.The third invention is characterized in that, in the first invention, phenol of 90 to 470 mg GAE (gallic acid equivalent), 1,100 to 1,800 mg of flavonoid and 18 to 35 mg of ascorbic acid are further contained in 100 mg of the root extract do.
제 4발명은, 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 제조방법에 관한 것으로, 이를 위해 청미래덩굴 뿌리를 시료를 직사광선을 피하여 그늘진 곳에서 1주 내지 3주 동안 자연 건조하는 S10단계;와, 상기 시료를 분쇄하여 추출 용매를 혼합하는 S20단계;와, 상기 시료를 환류시켜서 건조추출물을 추출하는 S30단계;와, 상기 건조추출물을 15℃ ~ 30℃로 냉각시킨 후 여과하여 여과추출물을 생성하는 S40단계; 및 상기 여과추출물을 감압 농축시켜 뿌리추출물을 추출하는 S50단계;를 포함하되, 상기 S50단계는 상기 여과추출물을 40℃ ~ 60℃ 범위 내에서 감압 농축시켜 10 ~ 350㎍/㎖의 농도로 뿌리추출물을 추출하고, 상기 뿌리추출물의 농도는 파골세포(osteoclast)의 생존율에 영향을 주지 않으면서, Cal-R(칼시토닌 수용체)의 단백질 및 TRAP(주석산염 저항산성 인산분해효소)의 단백질로 이루어진 군으로부터 선택되는 1종 이상의 발현을 감소시켜 골다공증 예방 및 개선에 사용되는 것을 특징으로 한다.A fourth aspect of the present invention relates to a method for preparing a root composition for prevention and improvement of osteoporosis, comprising the steps of: (a) A step S30 of extracting the dried extract by refluxing the sample, a step S30 of filtering the dried extract at a temperature of 15 to 30 占 폚 to produce a filtered extract, step; And extracting the root extract by concentrating the filtrate under reduced pressure. In step S50, the filtrate is concentrated under reduced pressure at 40 to 60 ° C to obtain a root extract And the concentration of the root extract was determined from the group consisting of proteins of Cal-R (calcitonin receptor) and TRAP (tartrate-resistant acid phosphatase) without affecting the survival rate of osteoclast And is used for prevention and improvement of osteoporosis by decreasing the expression of at least one selected.
제 5발명은, 제 4발명에서, 상기 S20단계는, 상기 추출 용매는 물 및 에탄올 농도가 60% ~ 80%인 에탄올 수용액 중에서 선택된 1종 또는 2종을 혼합한 것을 특징으로 한다.The fifth aspect of the present invention is the method according to the fourth aspect of the invention, wherein the extraction solvent is one or two selected from the group consisting of water and an aqueous ethanol solution having an ethanol concentration of 60% to 80%.
제 6발명은, 제 4발명에서, 상기 S20단계는, 상기 분쇄된 시료와 추출 용매는 100g : 800 ~ 1,100㎖의 비율로 혼합되는 것을 특징으로 한다.In a sixth aspect of the invention, in the fourth invention, the pulverized sample and the extraction solvent are mixed at a ratio of 100 g: 800 to 1,100 ml.
삭제delete
본 발명의 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물 및 이의 제조방법에 따르면,부작용을 최소화시키면서 파골세포 활성의 억제를 통하여 골다공증 예방 및/또는 개선 효과를 극대화 시킬 수 있는 효과가 있다.According to the present invention, it is possible to maximize the effect of preventing and / or improving osteoporosis through inhibition of osteoclast activity while minimizing side effects, according to the cheonggyeong vine root composition for prevention and improvement of osteoporosis.
또한 청미래덩굴 뿌리추출물에 페놀, 플라보노이드 및 아스코르브산을 포함시켜 항산화 성분을 일상생활에서 식용으로 섭취 할 수 있는 효과가 있다.In addition, phenol, flavonoid, and ascorbic acid are included in the root extract of Cheongmyeongrae, so that antioxidant components can be ingested in daily life.
도 1은 본 발명에 따른 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 제조방법에 따른 순서도,
도 2는 본 발명에 따른 청미래덩굴 뿌리조성물의 MTT assay를 통한 세포 독성 평가를 나타낸 그래프,
도 3은 본 발명에 따른 청미래덩굴 뿌리조성물의 TRAP 활성 평가를 나타낸 그래프,
도 4는 본 발명에 따른 청미래덩굴 뿌리조성물의 RANKL에 의해 유도되는 파골세포의 TRAP 및 Cal-R 단백질 발현양 이미지와 평가를 나타낸 그래프,
도 5는 청미래덩굴 뿌리추출물 처리에 따른 TRAP 염색에 의한 파골세포 양성세포 광학현미경 이미지 사진이다.FIG. 1 is a flow chart of a method for manufacturing a cheongyongra japonica root composition for prevention and improvement of osteoporosis according to the present invention,
FIG. 2 is a graph showing the cytotoxicity evaluation through the MTT assay of the Cheongmyeong roots composition according to the present invention,
FIG. 3 is a graph showing the TRAP activity evaluation of the Cheongmaia vine root composition according to the present invention,
FIG. 4 is a graph showing the image and evaluation of TRAP and Cal-R protein expression levels of RANKL-induced osteoclasts in the Cheongmaia vine root composition according to the present invention,
FIG. 5 is an image of an osteoclast-positive cell optical microscope image obtained by TRAP staining according to the treatment of Root Extract of Cheongpyeong Vine.
이하의 본 발명의 목적들, 다른 목적들, 특징들 및 이점들은 첨부된 도면과 관련된 이하의 바람직한 실시예들을 통해서 쉽게 이해될 것이다. 그러나 본 발명은 여기서 설명되는 실시예들에 한정되지 않고 다른 형태로 구체화될 수도 있다.BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, and advantages of the present invention will become more readily apparent from the following description of preferred embodiments with reference to the accompanying drawings. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms.
오히려, 여기서 소개되는 실시예들은 개시된 내용이 철저하고 완전해질 수 있도록 그리고 당업자에게 본 발명의 사상이 충분히 전달될 수 있도록 하기 위해 제공되는 것이다.Rather, the embodiments disclosed herein are provided so that the disclosure can be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
여기에 설명되고 예시되는 실시예들은 그것의 상보적인 실시예들도 포함한다.The embodiments described and exemplified herein also include their complementary embodiments.
본 명세서에서, 단수형은 문구에서 특별히 언급하지 않는 한 복수형도 포함한다. 명세서에서 사용되는 '포함한다(comprise)' 및/또는 '포함하는(comprising)'은 언급된 구성요소는 하나 이상의 다른 구성요소의 존재 또는 추가를 배제하지 않는다.In the present specification, the singular form includes plural forms unless otherwise specified in the specification. The terms "comprise" and / or "comprising" used in the specification do not exclude the presence or addition of one or more other elements.
이하, 도면을 참조하여 본 발명을 상세히 설명하도록 한다. 아래의 특정 실시예들을 기술하는데 있어서, 여러가지의 특정적인 내용들은 발명을 더 구체적으로 설명하고 이해를 돕기 위해 작성되었다. 하지만 본 발명을 이해할 수 있을 정도로 이 분야의 지식을 갖고 있는 독자는 이러한 여러 가지의 특정적인 내용들이 없어도 사용될수 있다는 것을 인지할 수 있다. 어떤 경우에는, 발명을 기술하는 데 있어서 흔히 알려졌으면서 발명과 크게 관련 없는 부분들은 본 발명을 설명하는 데 있어 혼돈을 막기 위해 기술하지 않음을 미리 언급해 둔다.Hereinafter, the present invention will be described in detail with reference to the drawings. In describing the specific embodiments below, various specific details have been set forth in order to provide a more detailed description of the invention. However, it will be appreciated by those skilled in the art that the present invention may be understood by those skilled in the art without departing from such specific details. In some cases, it should be mentioned in advance that it is common knowledge in describing an invention that parts not significantly related to the invention are not described in order to avoid confusion in explaining the present invention.
이하에서는 본 발명에 따른 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물에 관하여 설명하기로 한다.Hereinafter, the present invention will be described with reference to the present invention.
본 발명은 물 또는 에탄올수용액 중 어느 하나를 추출 용매로 하여 약학적으로 유효한 청미래덩굴 뿌리추출물로 이루어지되, 상기 청미래덩굴 뿌리추출물은 10 ~ 350㎍/㎖의 농도를 갖는 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물에 관한 것이다.The present invention relates to a medicinal composition for the prevention and treatment of osteoporosis, which has a concentration of 10 ~ 350 / / ml, and which comprises a phytotoxic root extract, which is pharmaceutically effective, using either water or an aqueous solution of ethanol as an extraction solvent. Roots composition.
여기서 상기 뿌리추출물의 농도는 파골세포(osteoclast)의 생존율에 영향을 주지 않으면서, Cal-R(칼시토닌 수용체)의 단백질 및 TRAP(주석산염 저항산성 인산분해효소)의 단백질로 이루어진 군으로부터 선택되는 1종 이상의 발현을 감소시켜 골다공증 예방 및 개선에 사용될 수 있다.Wherein the concentration of the root extract is selected from the group consisting of a protein of Cal-R (calcitonin receptor) and a protein of TRAP (tartrate-resistant acid phosphatase) without affecting the survival rate of osteoclast. And can be used for preventing and improving osteoporosis.
본 발명의 일실시예로서, 청미래덩굴 뿌리조성물은 뿌리추출물이 25㎍/㎖ 농도일 때, TRAP(주석산염 저항산성 인산분해효소)의 단백질 활성이 20% ~ 40%일 수 있다.In one embodiment of the present invention, the cheongmyeong vine root composition may have a protein activity of TRAP (tartrate-resistant acid phosphatase) of 20% to 40% when the root extract has a concentration of 25 / / ml.
본 발명의 또 다른 일실시예로서, 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 뿌리추출물은 물을 추출 용매로 추출한 경우, 100㎍/㎖ 농도일 때, TRAP(주석산염 저항산성 인산분해효소)의 단백질 활성이 50.0% ~ 59.0%이고, 250㎍/㎖ 농도일 때, TRAP(주석산염 저항산성 인산분해효소)의 단백질 활성이 20% ~ 28%일 수 있다.As another embodiment of the present invention, the root extract of the Cheonggyejung roots composition for prevention and improvement of osteoporosis is characterized in that when water is extracted with an extraction solvent, TRAP (tartrate-resistant acid phosphatase) at 100 μg / The protein activity of the TRAP (tartrate-resistant acid phosphatase) may be 20% to 28% when the protein activity is 50.0% to 59.0% and the concentration is 250 μg / ml.
본 발명의 바람직한 일실시예로서, 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 뿌리추출물은 에탄올을 추출 용매로 추출한 경우, 100㎍/㎖ 농도일 때, TRAP의 단백질 활성이 40.0% ~ 49.0%이고, 250 ㎍/㎖ 농도일 때, TRAP 효소 활성이 32% ~ 40%일 수 있다.As a preferred embodiment of the present invention, when extracting ethanol with an extraction solvent, the root extract of Cheongmaia vine root composition for prevention and improvement of osteoporosis has a protein activity of TRAP of 40.0% ~ 49.0% at a concentration of 100 / / ml, At a concentration of 250 μg / ml, the TRAP enzyme activity may be between 32% and 40%.
본 발명의 바람직한 일실시예로서, 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 뿌리추출물이 250㎍/㎖ 농도일 때, TRAP의 단백질 활성 발현을 55% ~ 80% 억제시킬 수 있다.As a preferred embodiment of the present invention, the expression of TRAP protein activity can be inhibited by 55% to 80% at a concentration of 250 μg / ml of the root extract of the Cheonggyeonggwon root composition for osteoporosis prevention and improvement.
본 발명의 바람직한 일실시예로서, 청골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 뿌리추출물은 물을 추출 용매로 추출한 경우, 250㎍/㎖ 농도일 때, TRAP의 단백질 활성 발현을 70.0% ~ 80.0% 억제 시킬 수 있다.As a preferred embodiment of the present invention, when water was extracted with an extraction solvent, the root extract of Cheonggyejung roe composition for prevention and improvement of blue osteoporosis showed 70.0% ~ 80.0% of TRAP protein activity at a concentration of 250 / / .
본 발명의 바람직한 일실시예로서, 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물은 에탄올을 추출 용매로 추출한 경우, 250㎍/㎖ 농도일 때, TRAP의 단백질 활성 발현을 55.0% ~ 65.0% 억제시킬 수 있다.
본 발명의 바람직한 일실시예로서, 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 뿌리추출물은 250㎍/㎖ 농도일 때, Cal-R(칼시토닌 수용체)의 단백질 발현을 16% ~ 40% 억제 시킬 수 있다.In one preferred embodiment of the present invention, when the extract of ethanol is extracted with a solvent at a concentration of 250 / / ml, the expression of TRAP protein activity can be inhibited by 55.0% ~ 65.0% .
As a preferred embodiment of the present invention, the root extract of Cheonggyeong vine root composition for prevention and improvement of osteoporosis can inhibit protein expression of Cal-R (calcitonin receptor) by 16% to 40% at a concentration of 250 / / ml .
삭제delete
본 발명의 바람직한 일실시예로서, 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 뿌리추출물은 물을 추출 용매로 추출한 경우, 250㎍/㎖ 농도일 때, Cal-R(칼시토닌 수용체)의 단백질 발현을 18.0% ~ 30.0% 억제 시킬 수 있다.As a preferred embodiment of the present invention, when water was extracted with an extraction solvent, the root extract of Cheongmaia vine root composition for prevention and improvement of osteoporosis showed protein expression of Cal-R (calcitonin receptor) at 18.0 % ~ 30.0%.
본 발명의 바람직한 일실시예로서, 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 뿌리추출물은 에탄올을 추출 용매로 추출한 경우, 250㎍/㎖ 농도일 때, Cal-R(칼시토닌 수용체)의 단백질 발현을 16.0% ~ 40.0% 억제시킬 수 있다.As a preferred embodiment of the present invention, the root extract of the cheonggyejung roots composition for prevention and improvement of osteoporosis showed protein expression of Cal-R (calcitonin receptor) at a concentration of 250 μg / ml when extracting ethanol with an extraction solvent at 16.0 % ~ 40.0%.
본 발명의 바람직한 일실시예로서, 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 뿌리추출물은 마우스 대식 세포주 RAW246.7세포를 이용하여 RANKL(Receptor activator of nuclear fractor-κB ligand)에 의해 유도된 파골세포 생존율 측정시, 250㎍/㎖ 농도일 때, 파골세포 생존율이 92.0% ~ 128%일 수 있다.As a preferred embodiment of the present invention, the root extract of the Cheonggyeong vine root composition for prevention and improvement of osteoporosis was prepared by using RANKL (Receptor activator of nuclear factor-κB ligand) induced osteoclast survival rate using mouse macrophage RAW246.7 cells When measured at a concentration of 250 μg / ml, the osteoclast survival rate may be 92.0% to 128%.
본 발명의 바람직한 일실시예로서, 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 뿌리추출물은 물을 추출 용매로 추출한 경우, 250㎍/㎖ 농도일 때, 파골세포 생존율이 92% ~ 106%일 수 있다.As a preferred embodiment of the present invention, the root extract of Rootgrass roots composition for prevention and improvement of osteoporosis can have an osteoclast survival rate of 92% ~ 106% when water is extracted with an extraction solvent at a concentration of 250 / / ml .
본 발명의 바람직한 일실시예로서, 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 뿌리추출물은 에탄올을 추출용매로 추출한 경우, 250㎍/㎖ 농도일 때, 파골세포 생존율이 102% ~ 128%일 수 있다.As a preferred embodiment of the present invention, the roots extract of Cheongmaia vine root composition for prevention and improvement of osteoporosis can have an osteoclast survival rate of 102% ~ 128% when ethanol is extracted with an extraction solvent at a concentration of 250 / / ml .
또한 본원발명의 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물은 상기 뿌리추출물 100mg에 대하여, 90 ~ 470mg GAE(gallic acid equivalent)의 페놀과, 1,100 ~ 1,800mg의 플라보노이드와, 18 ~ 35mg의 아스코르브산이 더 포함되어 항산화 효과를 갖도록 구성될 수 있다.In addition, the cheonggyeong vine root composition for prevention and improvement of osteoporosis according to the present invention further comprises phenol of 90 to 470 mg GAE (gallic acid equivalent), 1,100 to 1,800 mg of flavonoid and 18 to 35 mg of ascorbic acid per 100 mg of the root extract And can be configured to have an antioxidative effect.
좀 더 구체적으로는 상기 뿌리추출물이 물을 추출 용매로 추출한 경우, 뿌리추출물의 100mg에 대하여, 페놀 함량이 458 ~ 462 mg GAE(gallic acid equivalent)이고, 플라보노이드 함량이 1,103 ~ 1,116 mg이며, 아스코르브산 함량이 19 ~ 21 mg을 포함할 수 있다.More specifically, when the root extract is extracted with an extraction solvent, the phenol content is 458 to 462 mg GAE (gallic acid equivalent), the flavonoid content is 1,103 to 1,116 mg, and the ascorbic acid The content may include 19 to 21 mg.
그리고 상기 뿌리추출물이 에탄올을 추출 용매로 추출한 경우, 뿌리추출물의 100mg에 대하여, 페놀 함량이 90 ~ 92 mg GAE(gallic acid equivalent)이고, 총 플라보노이드 함량이 1,743 ~ 1,753 mg이며, 아스코르브산 함량이 28 ~ 30 mg을 포함할 수 있다.When the extract of the root is extracted with ethanol, the phenol content is 90 to 92 mg of GAE (gallic acid equivalent), the total flavonoid content is 1,743 to 1,753 mg, and the ascorbic acid content is 28 To 30 mg.
이와 같이, 본 발명의 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물은 페놀, 플라보노이드 및 아스코르빈산을 고함량으로 포함하는 바, 항산화 효과가 우수하면서도 TRAP 효소의 활성 즉, 파골세포 분화 활성 억제를 통한 골다공증 예방, 개선 효과가 우수하다.As described above, the cheonggyeong vine root composition for prevention and improvement of osteoporosis according to the present invention contains phenol, flavonoid and ascorbic acid in a high content, and it has excellent antioxidative effect, but also has activity of TRAP enzyme, that is, osteoporosis Prevention, and improvement.
이하에서는 본 발명에 따른 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 제조방법에 관하여 첨부되어진 도면과 함께 더불어 상세히 설명하기로 한다.BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a method for preparing a cheonggyeong vine root composition for prevention and improvement of osteoporosis according to the present invention will be described in detail with reference to the accompanying drawings.
도 1과 같이, 먼저 S10단계에서는 청미래덩굴 뿌리를 시료를 직사광선을 피하여 그늘진 곳에서 1주 내지 3주 동안 자연 건조한다. As shown in Fig. 1, first, in step S10, the roots of Cheongmaia vine are naturally dried for 1 week to 3 weeks in a shady place avoiding direct sunlight.
S20단계에서는 상기 시료를 분쇄하여 추출 용매를 혼합한다.In step S20, the sample is pulverized and the extraction solvent is mixed.
이 때 S20단계에서 추출 용매는 물 및 에탄올 수용액 중에서 선택된 1종 또는 2종을 혼합하여 사용할 수 있다.At this time, in step S20, the extraction solvent may be a mixture of one or two selected from water and an aqueous solution of ethanol.
바람직하게는 추출 용매로서 에탄올 수용액을 사용하되, 에탄올 농도가 60% ~ 80%이 되도록 사용하는 것이 추출 효과 면에서 유리하기 때문이다.It is preferable to use an ethanol aqueous solution as an extraction solvent, and to use ethanol so that the ethanol concentration is 60% to 80% in terms of extraction effect.
또한 S20단계에서 시료와 추출 용매는 100g : 800 ~ 1,100㎖ 의 비율로 혼합되는 것이 바람직하다.In step S20, it is preferable that the sample and the extraction solvent are mixed at a ratio of 100 g: 800 to 1,100 ml.
여기서 추출 용매가 800㎖ 미만이면 시료와 추출 용매의 혼합이 불완전하게 되어 유효성분의 추출이 충분하지 않을 수 있고, 1,200㎖ 를 초과하면 일정 온도를 유지하며 뿌리추출물을 추출하는 방법에 문제가 있을 수 있기 때문에 바람직하지 않다.If the extraction solvent is less than 800 ml, the mixing of the sample and the extraction solvent may become incomplete and the extraction of the active ingredient may not be sufficient. If the extraction solvent is more than 1,200 ml, there may be a problem in the method of extracting the root extract Which is not desirable.
아울러 S30단계에서는 시료를 환류시켜서 건조추출물을 추출한다.In step S30, the sample is refluxed to extract the dried extract.
여기서 환류는 80℃ ~ 95℃ 하에서, 바람직하게는 82℃ ~ 88℃ 하에서, 2.5 ~ 3.5시간씩 3번 ~ 5번 수행할 수 있다.Here, the reflux can be carried out at 80 ° C to 95 ° C, preferably 82 ° C to 88 ° C, for 3 to 5 times for 2.5 to 3.5 hours.
S40단계에서는 상기 건조추출물을 15℃ ~ 30℃로 냉각시킨 후 여과하여 여과추출물을 생성한다.In step S40, the dried extract is cooled to 15 ° C to 30 ° C and filtered to produce a filtered extract.
S50단계에서는 상기 여과추출물을 감압 농축시켜 뿌리추출물을 추출하여 완성한다.In step S50, the filtrate is concentrated under reduced pressure to extract the root extract.
이 때 S50단계에서 여과추출물을 40℃ ~ 60℃ 범위 내에서 감압 농축시켜 10 ~ 350㎍/㎖의 농도로 뿌리추출물을 추출할 수 있다.At this time, in step S50, the filtrate extract can be concentrated under reduced pressure in the range of 40 ° C to 60 ° C to extract the root extract at a concentration of 10 to 350 μg / ml.
여기서 40℃ 미만에서 감압농축을 수행하면 농축의 시간이 장시간이 될 수 있는 문제가 있을 수 있고, 60℃를 초과하면 유효 성분의 변성이 있을 수 있는 문제가 있을 수 있기 때문에 바람직하지 않다.Concentration at a reduced pressure of less than 40 캜 may cause a problem that the time for concentration may be prolonged, and if it exceeds 60 캜, there is a possibility that the active ingredient may be denatured.
또한 뿌리추출물의 농도가 10 ㎍/㎖ 미만이면 파골세포효소 활성 저하 효과가 미비하여 골다공증 효과를 볼 수 없을 수 있고, 350㎍/㎖의 농도를 초과하면 독성에 의한 세포 생존율을 저하시키는 문제가 있을 수 있으므로 상기 범위 내의 농도로 뿌리추출물을 포함하는 것이 좋다.
여기서 상기 뿌리추출물의 농도는 파골세포(osteoclast)의 생존율에 영향을 주지 않으면서, Cal-R(칼시토닌 수용체)의 단백질 및 TRAP(주석산염 저항산성 인산분해효소)의 단백질로 이루어진 군으로부터 선택되는 1종 이상의 발현을 감소시켜 골다공증 예방 및 개선에 사용될 수 있다.If the concentration of the root extract is less than 10 / / ㎖, osteoporosis effect can not be obtained due to insufficient osteoclast enzyme activity lowering effect. If the concentration is more than 350 / / ㎖, there is a problem of lowering cell survival rate due to toxicity It is preferable to include the root extract at a concentration within the above range.
Wherein the concentration of the root extract is selected from the group consisting of a protein of Cal-R (calcitonin receptor) and a protein of TRAP (tartrate-resistant acid phosphatase) without affecting the survival rate of osteoclast. And can be used for preventing and improving osteoporosis.
실시예1: 물을 추출 용매로 하여 감압 농축하여 청미래덩굴 뿌리조성물을 제조하였다.Example 1: Water was used as an extraction solvent and concentrated under reduced pressure to prepare a root-knot root composition.
실시예2: 70% 농도의 에타올 수용액을 추출 용매로 하여 청미래덩굴 뿌리조성물를 제조하였다.Example 2: Cheongmyong vine root composition was prepared by using a 70% aqueous solution of EtOH aqueous solution as an extraction solvent.
[실험예 1] 폴리페놀, 플라보노이드 및 아스코르빈산 함량 측정[Experimental Example 1] Measurement of polyphenol, flavonoid and ascorbic acid content
실시예1 및 실시예2의 냉동고에 보관된 청미래덩굴 뿌리조성물 100g에 함유된 폴리페놀, 플라보노이드 및 아스코르빈산의 함량을 각각 측정하였다.The content of polyphenols, flavonoids and ascorbic acid contained in 100 g of the Cheonggyeonggwina root composition stored in the freezer of each of Example 1 and Example 2 was measured.
이 때, 폴리페놀과 플라보노이드 함량 측정을 위한 폴리-시오칼토 시약(FolinCiocalteau reagent), 갈산(gallic acid), 아질산 나트륨(sodium nitrite), 카테킨(catechin), 트라이클로로아세트산(trichloroacetic acid), 아스코르빈산(ascorbic acid)을 사용하였다.At this time, the polyphenol and flavonoid content were measured using FolinCiocalteau reagent, gallic acid, sodium nitrite, catechin, trichloroacetic acid, ascorbic acid, (ascorbic acid) was used.
그리고 폴리페놀은 갈산으로 정량하여 건조함량 당 100g 기준으로 측정하였고, 플라보노이드 함량은 카테킨으로 정량하여 건조함량 당 100g 기준으로 측정하였으며, 아스크로빈산 함량은 아스코르빈산으로 정량하여 건조함량 당 100g 기준으로 측정하였다. 측정값은 mg 단위로 표기하였다.The amount of flavonoid was measured by catechin and the amount of ascorbic acid was determined by ascorbic acid and the amount of ascorbic acid was measured by 100 g per dry content . The measured values are expressed in mg units.
(mg GAE1 )/100g of dry mass)Total phenol content
(mg GAE 1 ) / 100 g of dry mass)
(mg CE2)/100g of dry mass)Total flavonoid content
(mg CE 2) / 100 g of dry mass)
(mg/100g of dry mass)Ascorbic acid content
(mg / 100 g of dry mass)
(%)yield
(%)
(SC-W)Example 1
(SC-W)
(SC-E)Example 2
(SC-E)
상기 [표 1]의 측정 결과를 살펴보면, 실시예1 및 실시예2를 각각 서로 비교해 보면, 페놀 함량은 에탄올 추출 용매보다 물 추출 용매가 상대적으로 높았고, 플라보노이드 및 아스크로빈산 함량은 물 추출 용매 보다 에탄올 추 출용매가 상대적으로 높은 결과를 보였다.Comparing the results of Table 1 with those of Example 1 and Example 2, phenol content was higher in water extracting solvent than ethanol extracting solvent, and flavonoid and ascorbic acid content were higher in water extracting solvent Ethanol extraction solvent was relatively higher than that of ethanol extract.
[실험예 2] 파골세포 생존율 측정 실험[Experimental Example 2] Measurement of osteoclast survival rate
실시예1 및 실시예2의 청미래덩굴 뿌리조성물 각각을 PBS (Dulbecco's, phosphate buffered saline)에 하기 [표 2]와 같은 농도로 희석하여 골다공증 예방 및 개선용 조성물을 각각 제조한 후, 하기와 같은 방법으로 파골세포의 생존율을 측정하였다.Each of the cheonggyeong roots composition of Example 1 and Example 2 was diluted with PBS (Dulbecco's, phosphate buffered saline) at the concentrations shown in Table 2 below to prepare compositions for osteoporosis prevention and improvement, The survival rate of osteoclasts was measured.
(1) 파골세포 배양(1) osteoclast culture
파골세포 분화 유도를 위한 마우스 대식 세포주 RAW264.7 세포는 ATCC(American Type Culture Collection; Rockville, MD, USA)로부터 구입하였으며, RAW264.7 세포는 10% FBS(fetal bovine serum, Gibco-BRL사)와 1% 페니실린-스트렙토마이신(penicillin-streptomycin, 100 U/㎖, 100 ㎍/㎖, Gibco-BRL사)이 포함된 DMEM 배지(Dulbecco’s modified Eagle’s medium)에 10% FBS와 1% 페니실린-스트렙토마이신을 첨가하여 5% CO2,37℃배양기(incubator)에서 계대 배양하였다.RAW264.7 cells were purchased from ATCC (American Type Culture Collection, Rockville, Md., USA) and RAW264.7 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Gibco-BRL) 10% FBS and 1% penicillin-streptomycin were added to DMEM medium (Dulbecco's modified Eagle's medium) containing 1% penicillin-streptomycin (100 U / ml, 100 쨉 g / ml, Gibco-BRL) And subcultured in a 5% CO 2 , 37 ° C incubator.
(2) 파골세포 생존율 측정(2) Measurement of osteoclast survival rate
하기 [표 2]의 농도를 갖는 추출물 각각의 RAW264.7 세포에 대한 생존 및 간접적 독성평가를 위하여 MTT assay를 시행하였다. 96-well plate에 1×104cells/well농도로 분주하여 DMEM에 10% FBS와 1% 페니실린-스트렙토마이신의 조건으로 24시간 동안 안정화시켰다. MTT assays were performed to assess the survival and indirect toxicity of RAW264.7 cells in extracts with the concentrations listed in Table 2 below. Cells were seeded at a density of 1 × 10 4 cells / well in a 96-well plate and stabilized in DMEM for 24 hours under conditions of 10% FBS and 1% penicillin-streptomycin.
다음으로, 파골세포 분화 유도를 위하여 10% FBS와 1% 페니실린-스트렙토마이신이 함유된 α-MEM에 RANKL(100 ng/㎖)을 첨가한 뒤, 하기 [표 2]의 농도를 갖도록 실시예1 및 실시예2의 청미래덩굴 뿌리조성물로 배양 처리하였다. 그리고, 각 뿌리조성물을 처리하지 않은 군을 대조군으로 설정하였다. Next, RANKL (100 ng / ml) was added to? -MEM containing 10% FBS and 1% penicillin-streptomycin for the induction of osteoclast differentiation, And the Crimea vine root composition of Example 2. The groups not treated with each root composition were set as a control group.
배양 3일 후 MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)용액 2㎎을 첨가하여 5% CO2,37℃배양기에서 2시간 동안 반응시킨 뒤 생성된 포마잔 결정(formazan crystals)을 DMSO(dimetyl sulfoxide)로 용해하였다. After 3 days of culture, 2 mg of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) solution was added and reacted in a 5% CO 2 incubator at 37 ° C for 2 hours. Formazan crystals were dissolved in DMSO (dimethyl sulfoxide).
그리고 이를 ELISA reader(BIO-RAD 450, USA)를 이용하여 540nm에서 흡광도를 확인하였고, 그 결과를 하기 [표 2] 및 도 2에 나타내었다.The absorbance was confirmed at 540 nm using an ELISA reader (BIO-RAD 450, USA). The results are shown in Table 2 and FIG. 2, respectively.
(SC-W)Example 1
(SC-W)
(SC-E)Example 2
(SC-E)
상기 [표 2] 및 도 2의 그래프를 살펴보면, 마우스 대식 세포주 RAW264.7 세포를 이용하여 RANKL에 의해 유도된 파골세포 생존율이 청미래덩굴 뿌리조성물의 0 ~ 250㎍/㎖ 농도에 이르기까지 각 뿌리조성물의 농도가 증가할수록, 물 추출물(SC-W) 보다 에탄올 추출물(SC-E)의 파골세포의 생존율이 상대적으로 높은 경향을 보였다.2, the mouse macrophage cell line RAW264.7 cells were used to determine the survival rate of osteoclast cells induced by RANKL to a concentration of 0 to 250 μg / ml of Cheongmaia japonica root composition (SC-E) than that of water extract (SC-W), the survival rate of osteoclasts was relatively higher than that of water extract (SC-W).
그러나, 500㎍/㎖ 농도는 물 추출 용매는 56.84%, 에탄올 추출 용매는 53.49%의 세포 생존율을 보임으로써 세포독성을 가지는 것으로 판단된다.However, the concentration of 500 ㎍ / ㎖ was found to be cytotoxic by showing cell viability of 56.84% for water extraction solvent and 53.49% for ethanol extraction solvent.
[실험예 3] TRAP 효소 활성 측정 실험[Experimental Example 3] Measurement of TRAP enzyme activity
골세포 특이 지표인 TRAP 효소의 활성을 측정하기 위하여 TRAP solution assay를 Tintut 등 (2002)의 방법으로 수행하였다. TRAP 효소는 ATP와 니트로페닐 포스페이트(nitrophenyl phosphate)가 존재할 때 높은 활성을 가지고, 골 흡수작용시 분비가 증가되기 때문에 그 활성도 측정하여 파골세포 분화정도를 확인하는 데 이용할 수 있다.TRAP solution assay was performed by Tintut et al. (2002) to measure the activity of TRAP enzyme which is a specific osteocyte marker. The TRAP enzyme has high activity when ATP and nitrophenyl phosphate are present and can be used to confirm osteoclast differentiation level by measuring activity of osteoclast during bone resorption.
하기 [표 3]의 농도를 갖도록 실시예1 및 실시예2의 청미래덩굴 뿌리조성물로 처리한 3일된 세포와 분화제를 PBS로 수세 후 차가운 라이시스 버퍼(cold lysis buffer, 90mM 구연산염, pH4.8, 타르타르산나트륨 80mM 함유한 0.1% Triton X-100)를 80㎕씩 분주하여 10분간 처리하고, 20mM p-니트로페닐 포스페이트(p-nitrophenyl phosphate, substrate solution)을 80㎕씩 첨가하여 5% CO2,37℃ 배양기에서 20분간 반응시켰다. The 3-day-old cells and the differentiation agent treated with the Crimea vine root composition of Examples 1 and 2 were washed with PBS and then washed with cold lysis buffer (90 mM citrate, pH 4.8 And 80 μl of 20 mM p-nitrophenyl phosphate (substrate solution) were added to each well, followed by addition of 5% CO 2 , And incubated in a 37 ° C incubator for 20 minutes.
다음으로, 40㎕의 0.5 N NaOH로 반응을 중지시킨 다음, ELISA reader를 이용하여 405nm에서 흡광도를 확인하였고, 그 결과를 하기 [표 3] 및 도 3에 나타내었다.Next, the reaction was stopped with 40 μl of 0.5 N NaOH, and the absorbance was confirmed at 405 nm using an ELISA reader. The results are shown in Table 3 and FIG. 3.
(㎍/㎖)density
(占 퐂 / ml)
(SC-W)Example 1
(SC-W)
(SC-E)Example 2
(SC-E)
상기 [표 3] 도 3를 살펴보면, 실시예1,2의 TRAP 활성도는 농도 의존적으로 RANKL 만을 처리한 대조군에 비하여 현저하게 낮은 활성도를 보이는 경향이 있었다. 세포 치사 활성이 없는 250㎍/㎖ 농도에서 물 추출(SC-W, 22.14%) 및 에탄올 추출(SC-E, 37.18%)로서 대조군에 비해 현저하게 감소된 파골세포 분화 활성을 보였다. 특히, 실시예1의 물 추출 용매(SC-W)의 경우, 실시예12의 에탄올 추출 용매(SC-E) 보다 현저하게 낮은 TRAP 활성을 보여서, 가장 우수한 파골세포활성의 억제능을 갖는 것을 확인할 수 있었다.3, TRAP activity of Examples 1 and 2 tended to be significantly lower than that of the control group treated with RANKL only in a concentration-dependent manner. (SC-W, 22.14%) and ethanol extract (SC-E, 37.18%) at a concentration of 250 / / ml without lethal activity showed a significantly reduced osteoclast differentiation activity as compared with the control. In particular, the water extracting solvent (SC-W) of Example 1 showed significantly lower TRAP activity than the ethanol extracting solvent (SC-E) of Example 12, confirming that it has the best ability to inhibit osteoclast activity there was.
[실험예 4] TRAP 효소 및 Cal-R (칼시토닌 수용체) 단백질 발현 측정 실험[Experimental Example 4] Measurement of protein expression of TRAP enzyme and Cal-R (calcitonin receptor)
파골세포는 다양한 사이토카인, 호르몬 및 유전인자 조절 등으로 시작, 분화, 다핵화 그리고 성숙화 등 복잡한 과정에 의해 형성되는데, Cal-R(calcitonin receptor)와 TRAP이 존재하며 실제적으로 골 흡수작용을 하는 산 생성이 활발하고, acting ring을 형성하여 골 기질을 흡수한다고 알려져 있다. 이에 성숙 파골세포의 골기질 흡수 단계에 관여하는 TRAP 및 Cal-R(calcitonin receptor)단백질의 발현을 관찰하기 위하여 웨스턴블랏(western blot) 방법으로 수행하였다. Osteoclasts are formed by complex processes such as initiation, differentiation, polynucleation, and maturation of various cytokines, hormones and genetic factors. Cal-R (calcitonin receptor) and TRAP are present. And it is known to absorb the bone matrix by forming an acting ring. Western blot analysis was performed to observe the expression of TRAP and Cal-R (calcitonin receptor) proteins involved in bone morphogenesis in mature osteoclasts.
250㎍/㎖ 농도를 갖도록 실시예1 및 실시예2의 조성물로 처리한 3일된 세포와 분화제를 PBS로 수세 후 차가운 라이시스 버퍼(cold lysis buffer, Pro-PREPTM,iNiRON)를 4000㎕을 첨가하여 30분간 냉동(-20℃)처리하고, 원심분리(4℃, 13,000rpm, 10min)하여 상층액을 취하여 BCA 법으로 단백질을 정량하였다.The 3-day-old cells treated with the composition of Example 1 and Example 2 and the differentiation agent were washed with PBS to have a concentration of 250 μg / ml, and 4000 μl of cold lysis buffer (Pro-PREP ™ , iNiRON) The supernatant was collected by centrifugation (4 ° C, 13,000 rpm, 10 min), and protein was quantified by BCA method.
단백질 50㎍/㎖과 sample buffer(60 mM Tris-Cl; pH 6.8, 2% SDS, 25% glycerol, 14.4 mM 2-mercaptaethanol, 0.1% bromphenol blue)를 혼합하여 100℃에서 5분간 끓인 후 급속 냉각하였으며, 각 lane 별 120V에서 전기 영동하였다. PVDF막으로 단백질을 부착하였고, 5% bovine serum albumin로 blocking 하였으며, 1차 항체로 TRAP와 Cal-R을 각각 반응시킨 후, 2차 항체로 HRP-conjugated anti-rabbit으로 처리하였다.50 μg / ml of protein and 60 μl of sample buffer (pH 6.8, 2% SDS, 25% glycerol, 14.4 mM 2-mercaptaethanol, 0.1% bromphenol blue) were mixed and boiled at 100 ° C. for 5 minutes and then rapidly cooled And electrophoresed at 120V for each lane. Protein was attached to the PVDF membrane and blocked with 5% bovine serum albumin. The TRAP and Cal-R were reacted with the primary antibody and HRP-conjugated anti-rabbit was treated with the secondary antibody.
목적단백질을 동정하기 위하여 기질로 ECL(enhanced chemiluminescence)을 사용한 후, membrane을 X-ray film에 노출시켜 LAS3,000 image analyzer를 사용하여 밴드를 현상하여 정량화하였다. 그 결과를 하기 표 4 및 도 4에 나타내었다. To identify the target protein, ECL (enhanced chemiluminescence) was used as a substrate, and the band was developed and quantified using a LAS3000 image analyzer by exposing the membrane to an X-ray film. The results are shown in Table 4 and FIG.
[표 4] 및 도 4를 살펴보면, 청미래덩굴 뿌리조성물의 TRAP 단백질 발현은 대조군에 비하여 현저한 낮은 발현량을 보이는 경향이었다. 특히, 실시예1의 물 추출 용매(SC-W)의 경우, 실시예2의 에탄올 추출 용매(SC-E) 보다 현저하게 낮은 TRAP 단백질 발현을 보여서, 가장 우수한 파골세포활성의 억제능을 갖는 것을 확인할 수 있었다.As shown in [Table 4] and FIG. 4, TRAP protein expression of Cheongmaia vine root composition tended to show a significantly lower expression level than the control group. In particular, the water extracting solvent (SC-W) of Example 1 showed significantly lower TRAP protein expression than the ethanol extracting solvent (SC-E) of Example 2, confirming that it has the best ability to inhibit osteoclast activity I could.
또한, 청미래덩굴 뿌리조성물에서 Cal-R 단백질 발현은 대조군에 비하여 현저한 낮은 발현량을 보이는 경향이었고, 실시예1과 실시예2는 차이는 보이지 않았다.In addition, the expression of Cal-R protein in the cheonggyejagi root composition tended to show a significantly low expression level as compared with the control group, and there was no difference between Example 1 and Example 2.
따라서 RANKL에 의한 TRAP 효소활성의 억제와 마찬가지로 Cal-R와 TRAP의 단백질 발현 역시 청미래덩굴 뿌리조성물에 의해서 유의하게 억제된다는 것을 알 수 있었다.Therefore, it was found that the protein expression of Cal-R and TRAP was also significantly inhibited by the root composition of Cheongmyeonga, as well as the inhibition of TRAP enzyme activity by RANKL.
이는 청미래덩굴 뿌리조성물을 포함하는 유효성분이 NF-kB과 MAPKs 경로조절을 하고, NF-kB, NFATc1 그리고 c-Fos등을 조절하여 파골세포분화를 억제하는 것으로 판단될 수 있다.It can be concluded that the active ingredient including the cheonggyejagi root composition regulates the pathway of NF-kB and MAPKs and inhibits osteoclast differentiation by regulating NF-kB, NFATc1 and c-Fos.
그러므로 청미래덩굴 뿌리조성물은 파골세포분화 전사인자인 NFATc1을 억제하여 Cal-R와 TRAP를 감소시켰을 것으로 예상되고, 이로 인해 최종적으로 골기질 흡수가 억제될 것으로 판단될 수 있다.Therefore, it is anticipated that the rosemary roots composition would inhibit NFATc1, which is an osteoclast differentiation transcription factor, to reduce Cal-R and TRAP, thereby ultimately inhibiting bone resorption.
[실험예 5] TRAP 염색을 통한 양성파골세포 분화 관찰[Experimental Example 5] Observation of positive osteoclast differentiation by TRAP staining
RAW264.7 세포로부터 유도 분화된 파골세포에서 시그마 TRAP 염색(tartate resistant acid phosphate)을 통하여 성숙 파골세포의 형태를 광학현미경으로 관찰하고, 디지털 영상카메라로 이미지를 영상화하여 둥근 모양 형태의 파골세포(round-shaped osteoclast, ROC) 수를 관찰하였다.The morphology of mature osteoclasts was observed by optical microscope through sigma TRAP (Sigma TRAP) in osteoclast cells derived from RAW264.7 cells, and images were imaged with a digital image camera to determine the shape of round osteoclasts (round -shaped osteoclast, ROC) were observed.
실시예1 및 실시예2의 [표 1]에서 실시한 청미래덩굴 뿌리추출물 세포 치사 활성을 나타내지 않는 0 ~ 250㎍/㎖ 농도를 갖는 조성물로 각각 처리하여 3일간 배양한 후 TRAP 염색을 시행하였고, 그 결과를 도 5에 나타내었다.The cells were treated with a composition having a concentration of 0 to 250 μg / ml, which did not exhibit cell lethal activity, and the cells were cultured for 3 days, followed by TRAP staining. The results are shown in Fig.
도 5을 살펴보면, 대조군(control)에서 둥근 모양 형태 파골세포(ROC) 즉, 세포가 융합되면서 다핵의 성숙 파골세포가 형성되어 골 표면에 부착하여 골 흡수 작용을 하는 TRAP 양성 다핵형 파골세포가 뚜렷하게 관찰되었다. 5, when the round shaped osteoclast (ROC), that is, the cells were fused in the control (control), the osteoclasts were formed, and the osteoclast cells with TRAP-positive polynuclear osteoclasts were observed Respectively.
그리고, 대조군과 비교할 때, 모든 청미래덩굴 뿌리조성물의 처리군에서 현저하게 감소된 ROC와 융합되지 않은 단핵의 전파골세포와 불규칙 모양 형태 파골세포 (irregular shaped osteoclast, IOC)가 풍부하게 관찰되었다.In comparison with the control group, monocyte proliferating bone cells and irregular shaped osteoclasts (IOCs) unfused with ROC markedly reduced in all of the treatment groups of the Cheonggyejung root composition were abundantly observed.
도 5의 양성파골세포 분화 관찰을 통해서 RANKL에 의해 유도되는 파골세포 분화과정 중에 청미래덩굴 뿌리조성물의 농도에 의존적으로 파골세포 분화 억제에 직접적으로 관여한다는 것을 확인할 수 있었다.5 benign osteoclasts It was confirmed that RANKL-induced osteoclast differentiation was directly involved in inhibition of osteoclast differentiation depending on the concentration of Cheongmyeong roe root composition.
상기 [실험예1 ~ 5]를 통하여, 본 발명이 제시하는 방법으로 추출한 청미래덩굴 뿌리추출물이 파골세포 활성의 억제를 통해서 골다공증 예방 및/또는 개선 효과를 갖을 수 있음을 확인할 수 있었으며, 이러한 청미래덩굴 뿌리추출물을 이용하여 골다공증 예방 및/또는 개선용 의약품, 건강기능식품을 제공할 수 있음을 확인하였다.Through the above Experimental Examples 1 to 5, it was confirmed that the Root Extract of Cheongmaia japonica extracted by the method of the present invention can prevent and / or improve osteoporosis through inhibition of osteoclast activity, It was confirmed that the root extract can provide a drug for the prevention and / or improvement of osteoporosis and a health functional food.
상기 [실험예 1 ~ 5]의 모든 실험군은 3개 이상 수행하였으며 정량적 결과는 본 연구에 대한 실험결과는 통계분석 SAS 8.2를 이용하여 ANOVA (Analysis of variation) 검정을 실시한 후 Duncan's Multiple Range Test (DMRT)의 다중범위검정과 two-tailed Student’s t-test로 p < 0.05 수준에서 유의성을 검증하였고, 대조군에 대한 백분율로 평균 ± 표준편차로 나타냈다. In all of the experimental groups 1 to 5, three or more experiments were performed. The quantitative results were analyzed by ANOVA (Analysis of variation) using SAS 8.2, followed by Duncan's Multiple Range Test (DMRT ) And a two-tailed Student's t-test. The significance was tested at the p <0.05 level, and the mean ± SD was expressed as a percentage of the control group.
본 명세서에 기재된 실시예와 도면에 도시된 구성은 본 발명의 가장 바람직한 일 실시예에 불과할 뿐이고 본 발명의 기술적 사상을 모두 대변하는 것은 아니므로, 본 출원시점에 있어서 이들을 대체할 수 있는 다양한 균등물과 변형 예들이 있을 수 있음을 이해하여야 한다.It should be noted that the embodiments described in the present specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention and are not intended to represent all of the technical ideas of the present invention so that various equivalents And variations are possible.
Claims (7)
상기 청미래덩굴 뿌리추출물은 10 ~ 350㎍/㎖의 농도를 갖고,
상기 뿌리추출물의 농도는 파골세포(osteoclast)의 생존율에 영향을 주지 않으면서, Cal-R(칼시토닌 수용체)의 단백질 및 TRAP(주석산염 저항산성 인산분해효소)의 단백질로 이루어진 군으로부터 선택되는 1종 이상의 발현을 감소시켜 골다공증 예방 및 개선에 사용되는 것을 특징으로 하는 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물.
Wherein the extract is selected from the group consisting of water extracts and aqueous ethanol solutions,
The above-mentioned Cheongmyeongvin root extract has a concentration of 10 ~ 350 / / ml,
The concentration of the root extract may be selected from the group consisting of protein of Cal-R (calcitonin receptor) and protein of TRAP (tartrate-resistant acid phosphatase) without affecting the survival rate of osteoclast Wherein the composition is used for prevention and improvement of osteoporosis.
상기 뿌리추출물 100mg에 대하여, 90 ~ 470mg GAE(gallic acid equivalent)의 페놀과, 1,100 ~ 1,800mg의 플라보노이드와, 18 ~ 35mg의 아스코르브산이 더 포함되는 것을 특징으로 하는 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물.
The method according to claim 1,
Characterized in that 100 mg of the root extract further contains 90 to 470 mg of GAE (gallic acid equivalent) of phenol, 1,100 to 1,800 mg of flavonoid and 18 to 35 mg of ascorbic acid. .
상기 시료를 분쇄하여 추출 용매를 혼합하는 S20단계;
상기 시료를 환류시켜서 건조추출물을 추출하는 S30단계;
상기 건조추출물을 15℃ ~ 30℃로 냉각시킨 후 여과하여 여과추출물을 생성하는 S40단계; 및
상기 여과추출물을 감압 농축시켜 뿌리추출물을 추출하는 S50단계;를 포함하되,
상기 S50단계는 상기 여과추출물을 40℃ ~ 60℃ 범위 내에서 감압 농축시켜 10 ~ 350㎍/㎖의 농도로 뿌리추출물을 추출하고, 상기 뿌리추출물의 농도는 파골세포(osteoclast)의 생존율에 영향을 주지 않으면서, Cal-R(칼시토닌 수용체)의 단백질 및 TRAP(주석산염 저항산성 인산분해효소)의 단백질로 이루어진 군으로부터 선택되는 1종 이상의 발현을 감소시켜 골다공증 예방 및 개선에 사용되는 것을 특징으로 하는 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 제조방법.
Step S10 in which the roots of Cheongmyeira vine roots are naturally dried for 1 week to 3 weeks in a shady place avoiding direct sunlight;
A step S20 of pulverizing the sample and mixing the extraction solvent;
S30, in which the sample is refluxed to extract a dried extract;
Cooling the dried extract to a temperature of 15 ° C to 30 ° C and then filtering to produce a filtered extract; And
And extracting the root extract by concentrating the filtrate under reduced pressure,
In step S50, the filtrate extract is concentrated under reduced pressure at a temperature of 40 to 60 ° C to extract root extract at a concentration of 10 to 350 μg / ml. The concentration of the root extract affects the survival rate of osteoclast (Calcitonin receptor) and a protein of TRAP (tartrate-resistant acid phosphatase), and is used for prevention and improvement of osteoporosis by reducing the expression of at least one selected from the group consisting of Cal-R (calcitonin receptor) and TRAP A method for preparing a cheonggyejagi root composition for prevention and improvement of osteoporosis.
상기 S20단계는
상기 추출 용매는 물 및 에탄올 농도가 60% ~ 80%인 에탄올 수용액 중에서 선택된 1종 또는 2종을 혼합한 것을 특징으로 하는 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 제조방법.
5. The method of claim 4,
In step S20,
Wherein the extraction solvent is one or two selected from the group consisting of water and an ethanol aqueous solution having an ethanol concentration of 60% to 80%.
상기 S20단계는
상기 분쇄된 시료와 추출 용매는 100g : 800 ~ 1,100㎖ 의 비율로 혼합되는 것을 특징으로 하는 골다공증 예방 및 개선용 청미래덩굴 뿌리조성물의 제조방법.5. The method of claim 4,
In step S20,
Wherein the pulverized sample and the extraction solvent are mixed at a ratio of 100 g to 800 to 1,100 ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020160156090A KR101848171B1 (en) | 2016-11-22 | 2016-11-22 | Chinaroot Root Composition For The Prevention And Treatment Of Osteoporosis And Method Of Manufacturing The Same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020160156090A KR101848171B1 (en) | 2016-11-22 | 2016-11-22 | Chinaroot Root Composition For The Prevention And Treatment Of Osteoporosis And Method Of Manufacturing The Same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR101848171B1 true KR101848171B1 (en) | 2018-04-11 |
Family
ID=61975977
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020160156090A KR101848171B1 (en) | 2016-11-22 | 2016-11-22 | Chinaroot Root Composition For The Prevention And Treatment Of Osteoporosis And Method Of Manufacturing The Same |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR101848171B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20200088936A (en) * | 2019-01-15 | 2020-07-24 | 동신대학교산학협력단 | Composition for improvement of dental caries and periodontal disease using an extract of seeds of Phaseolus radiatus, etc. |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101086669B1 (en) * | 2010-09-15 | 2011-11-24 | (주)선바이오텍 | Method for preparing smilax china l and cosmetic compound for skin whitening compring extract from same |
-
2016
- 2016-11-22 KR KR1020160156090A patent/KR101848171B1/en active IP Right Grant
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101086669B1 (en) * | 2010-09-15 | 2011-11-24 | (주)선바이오텍 | Method for preparing smilax china l and cosmetic compound for skin whitening compring extract from same |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20200088936A (en) * | 2019-01-15 | 2020-07-24 | 동신대학교산학협력단 | Composition for improvement of dental caries and periodontal disease using an extract of seeds of Phaseolus radiatus, etc. |
| KR102269164B1 (en) | 2019-01-15 | 2021-12-13 | 동신대학교산학협력단 | Composition for improvement of dental caries and periodontal disease using an extract of seeds of Phaseolus radiatus, etc. |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Luo et al. | Green tea (Camellia sinensis) extract inhibits both the metastasis and osteolytic components of mammary cancer 4T1 lesions in mice | |
| KR101954325B1 (en) | Compositions and methods for managing or improving bone disorders, cartilage disorders, or both | |
| Qi et al. | Anthocyanin-rich extract from black rice (Oryza sativa L. Japonica) ameliorates diabetic osteoporosis in rats | |
| Shalan et al. | Noni leaf and black tea enhance bone regeneration in estrogen-deficient rats | |
| KR102049440B1 (en) | Composition for preventing and improving woman climacterium symptoms comprising extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume | |
| KR102334305B1 (en) | Composition for preventing or treating bon disease comprising extract of blueberry | |
| JP2022079461A (en) | Composition for remedying female climacteric syndrome symptoms | |
| KR101848171B1 (en) | Chinaroot Root Composition For The Prevention And Treatment Of Osteoporosis And Method Of Manufacturing The Same | |
| US20220323530A1 (en) | Composition for inhibiting osteoclasts containing agastache rugosa extract as active ingredient, and use thereof | |
| KR20080046543A (en) | Functional food comprising extract of nelumbo nucifera gaertn for the prevention and amelioration of osteoporosis | |
| KR101487761B1 (en) | Pharmaceutical Composition for the prevention or treatment of osteoporosis comprising Artemisinin | |
| EP1379146B1 (en) | Preparation for the prevention and/or treatment of altered bone metabolism | |
| KR20110007978A (en) | A composition for preventing and treating bone disease comprising colforsin daropate | |
| KR20150055667A (en) | Comoposition for Preventing or Treating Osteoporosis Comprising Extract Containing Scoparone Derived from Artemisia Capillaris | |
| KR101749974B1 (en) | Composition for the prevention and treatment of osteoporosis containing white Hibiscus syriacus L. flower extract | |
| KR101879398B1 (en) | Pharmaceutical composition for enhancing osteogenesis comprising garcinia mangostana extract and propolis extract | |
| TW200946125A (en) | Compositions and methods for maintaining bone health or reducing bone loss | |
| KR101254847B1 (en) | The composition of silibinin for treatment and prevention of osteoporosis | |
| KR101502465B1 (en) | A pharmaceutical composition comprising Alpinia Officinarum extracts for prevention and treatment of bone diseases or anti-vascular calcification activity | |
| KR20170015446A (en) | composition for the prevention and treatment ofosteoporosis containing Acyranthes bidentata Blume and ginseng extract | |
| KR20160051658A (en) | Composition for Effect of Protecting Liver Cells and Increasing Bone Density Using Germinated Fermented Soybean Extract Containing | |
| KR100752411B1 (en) | Extract of nelumbo nucifera gaertn having effect to prevent and treat osteoporosis | |
| KR101320974B1 (en) | Composition for treatment and prevention of bone diseases comprising extract of magnoliae flos | |
| KR101320975B1 (en) | Composition for treatment and prevention of bone diseases comprising extract of magnoliae flos's active components | |
| KR101791574B1 (en) | Pharmaceutical composition for preventing or treating female menopausal disorder comprising an extract of black rice aleurone layer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| GRNT | Written decision to grant |