KR100440863B1 - Extract of herb mixture for heamatopoiesis augmentation and protection from radiation - Google Patents
Extract of herb mixture for heamatopoiesis augmentation and protection from radiation Download PDFInfo
- Publication number
- KR100440863B1 KR100440863B1 KR10-2001-0028743A KR20010028743A KR100440863B1 KR 100440863 B1 KR100440863 B1 KR 100440863B1 KR 20010028743 A KR20010028743 A KR 20010028743A KR 100440863 B1 KR100440863 B1 KR 100440863B1
- Authority
- KR
- South Korea
- Prior art keywords
- cells
- radiation
- hematopoietic
- herbal
- extract
- Prior art date
Links
- 230000005855 radiation Effects 0.000 title claims abstract description 63
- 239000000284 extract Substances 0.000 title claims abstract description 10
- 239000000203 mixture Substances 0.000 title claims description 7
- 230000003416 augmentation Effects 0.000 title 1
- 239000012676 herbal extract Substances 0.000 claims abstract description 97
- 230000003394 haemopoietic effect Effects 0.000 claims abstract description 38
- 235000006484 Paeonia officinalis Nutrition 0.000 claims abstract description 28
- 230000036737 immune function Effects 0.000 claims abstract description 21
- 241001572175 Gaza Species 0.000 claims abstract description 16
- 235000001287 Guettarda speciosa Nutrition 0.000 claims abstract description 13
- 230000002708 enhancing effect Effects 0.000 claims abstract description 9
- 241000736199 Paeonia Species 0.000 claims description 27
- 241000411851 herbal medicine Species 0.000 claims description 20
- 241000125175 Angelica Species 0.000 claims description 12
- 239000008194 pharmaceutical composition Substances 0.000 claims description 10
- 239000004480 active ingredient Substances 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 39
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 15
- 238000011084 recovery Methods 0.000 abstract description 11
- 239000000126 substance Substances 0.000 abstract description 11
- 230000002265 prevention Effects 0.000 abstract description 9
- 231100000419 toxicity Toxicity 0.000 abstract description 8
- 230000001988 toxicity Effects 0.000 abstract description 8
- 201000010099 disease Diseases 0.000 abstract description 7
- 238000002156 mixing Methods 0.000 abstract description 6
- 210000000777 hematopoietic system Anatomy 0.000 abstract description 4
- 239000003173 antianemic agent Substances 0.000 abstract description 2
- 244000061520 Angelica archangelica Species 0.000 abstract 1
- 244000170916 Paeonia officinalis Species 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 52
- 210000004027 cell Anatomy 0.000 description 44
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 23
- 210000000952 spleen Anatomy 0.000 description 18
- 230000006870 function Effects 0.000 description 16
- 210000000601 blood cell Anatomy 0.000 description 15
- 210000002798 bone marrow cell Anatomy 0.000 description 15
- 210000000813 small intestine Anatomy 0.000 description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 210000002865 immune cell Anatomy 0.000 description 12
- 230000035755 proliferation Effects 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 210000004369 blood Anatomy 0.000 description 11
- 239000008280 blood Substances 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 11
- 230000000735 allogeneic effect Effects 0.000 description 10
- 210000000130 stem cell Anatomy 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 230000001965 increasing effect Effects 0.000 description 9
- 241000283074 Equus asinus Species 0.000 description 8
- 206010073306 Exposure to radiation Diseases 0.000 description 8
- 210000001185 bone marrow Anatomy 0.000 description 8
- 101100042788 Caenorhabditis elegans him-1 gene Proteins 0.000 description 7
- 230000024932 T cell mediated immunity Effects 0.000 description 7
- 208000035475 disorder Diseases 0.000 description 7
- 210000003743 erythrocyte Anatomy 0.000 description 7
- 208000018706 hematopoietic system disease Diseases 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 210000002536 stromal cell Anatomy 0.000 description 7
- 208000018522 Gastrointestinal disease Diseases 0.000 description 6
- 231100000111 LD50 Toxicity 0.000 description 6
- 230000004069 differentiation Effects 0.000 description 6
- 230000008929 regeneration Effects 0.000 description 6
- 238000011069 regeneration method Methods 0.000 description 6
- 230000006907 apoptotic process Effects 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 230000000968 intestinal effect Effects 0.000 description 5
- 210000004698 lymphocyte Anatomy 0.000 description 5
- 229930014626 natural product Natural products 0.000 description 5
- 230000001737 promoting effect Effects 0.000 description 5
- 210000004988 splenocyte Anatomy 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 241001494479 Pecora Species 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 235000013376 functional food Nutrition 0.000 description 4
- 208000024908 graft versus host disease Diseases 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 239000007928 intraperitoneal injection Substances 0.000 description 4
- 210000002540 macrophage Anatomy 0.000 description 4
- 239000000546 pharmaceutical excipient Substances 0.000 description 4
- 230000002062 proliferating effect Effects 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 210000000689 upper leg Anatomy 0.000 description 4
- 238000011740 C57BL/6 mouse Methods 0.000 description 3
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 3
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 3
- 208000032843 Hemorrhage Diseases 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 210000000628 antibody-producing cell Anatomy 0.000 description 3
- 230000001640 apoptogenic effect Effects 0.000 description 3
- 230000000740 bleeding effect Effects 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 230000005779 cell damage Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000007123 defense Effects 0.000 description 3
- 210000002744 extracellular matrix Anatomy 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000008616 guibi-tang Substances 0.000 description 3
- 208000026278 immune system disease Diseases 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000011065 in-situ storage Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 230000001172 regenerating effect Effects 0.000 description 3
- 239000009536 samultang Substances 0.000 description 3
- 230000003393 splenic effect Effects 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 150000003573 thiols Chemical class 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 210000004291 uterus Anatomy 0.000 description 3
- 241000208340 Araliaceae Species 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000016359 Fibronectins Human genes 0.000 description 2
- 108010067306 Fibronectins Proteins 0.000 description 2
- 239000012981 Hank's balanced salt solution Substances 0.000 description 2
- 238000011887 Necropsy Methods 0.000 description 2
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 2
- 235000003140 Panax quinquefolius Nutrition 0.000 description 2
- 208000019095 Radiation-induced disease Diseases 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 2
- 238000011047 acute toxicity test Methods 0.000 description 2
- 210000001789 adipocyte Anatomy 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000003125 aqueous solvent Substances 0.000 description 2
- 208000015322 bone marrow disease Diseases 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 235000014121 butter Nutrition 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 208000037887 cell injury Diseases 0.000 description 2
- 208000015114 central nervous system disease Diseases 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000005757 colony formation Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 235000008434 ginseng Nutrition 0.000 description 2
- 230000002489 hematologic effect Effects 0.000 description 2
- 230000002949 hemolytic effect Effects 0.000 description 2
- 230000002519 immonomodulatory effect Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 239000002955 immunomodulating agent Substances 0.000 description 2
- 229940121354 immunomodulator Drugs 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 210000003134 paneth cell Anatomy 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 102000003390 tumor necrosis factor Human genes 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 239000000080 wetting agent Substances 0.000 description 2
- AZFYOKHCKZPHNU-JJJJRQIZSA-N 2-aminoethyl carbamimidothioate;[[(2r,3s,4r)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound NCCSC(N)=N.C1=NC=2C(N)=NC=NC=2N1C1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O AZFYOKHCKZPHNU-JJJJRQIZSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 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
- 206010003445 Ascites Diseases 0.000 description 1
- 208000031229 Cardiomyopathies Diseases 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 241000222336 Ganoderma Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 206010022653 Intestinal haemorrhages Diseases 0.000 description 1
- 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 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 240000001439 Opuntia Species 0.000 description 1
- 235000013389 Opuntia humifusa var. humifusa Nutrition 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 206010038460 Renal haemorrhage Diseases 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 229920002472 Starch Polymers 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
- 229930006000 Sucrose Natural products 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 244000299461 Theobroma cacao Species 0.000 description 1
- 235000005764 Theobroma cacao ssp. cacao Nutrition 0.000 description 1
- 235000005767 Theobroma cacao ssp. sphaerocarpum Nutrition 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 231100000403 acute toxicity Toxicity 0.000 description 1
- 230000007059 acute toxicity Effects 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- LNQVTSROQXJCDD-UHFFFAOYSA-N adenosine monophosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)C(OP(O)(O)=O)C1O LNQVTSROQXJCDD-UHFFFAOYSA-N 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001147 anti-toxic effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 235000001046 cacaotero Nutrition 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000001100 crypt cell Anatomy 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 235000012888 dietary physiology Nutrition 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- IZEKFCXSFNUWAM-UHFFFAOYSA-N dipyridamole Chemical compound C=12N=C(N(CCO)CCO)N=C(N3CCCCC3)C2=NC(N(CCO)CCO)=NC=1N1CCCCC1 IZEKFCXSFNUWAM-UHFFFAOYSA-N 0.000 description 1
- 229960002768 dipyridamole Drugs 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 208000001780 epistaxis Diseases 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- -1 ethyl oleate 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
- 238000000605 extraction Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000005251 gamma ray Effects 0.000 description 1
- 230000002496 gastric effect Effects 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
- 239000008187 granular material Substances 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 229960002706 gusperimus Drugs 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002434 immunopotentiative effect Effects 0.000 description 1
- 229960001438 immunostimulant agent Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- VMPHSYLJUKZBJJ-UHFFFAOYSA-N lauric acid triglyceride Natural products CCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCC)COC(=O)CCCCCCCCCCC VMPHSYLJUKZBJJ-UHFFFAOYSA-N 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 229960003511 macrogol Drugs 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- IDINUJSAMVOPCM-INIZCTEOSA-N n-[(1s)-2-[4-(3-aminopropylamino)butylamino]-1-hydroxy-2-oxoethyl]-7-(diaminomethylideneamino)heptanamide Chemical compound NCCCNCCCCNC(=O)[C@H](O)NC(=O)CCCCCCN=C(N)N IDINUJSAMVOPCM-INIZCTEOSA-N 0.000 description 1
- 208000011309 nasal bleeding Diseases 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000006186 oral dosage form Substances 0.000 description 1
- 230000005813 organ abnormality Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 208000021090 palsy Diseases 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000006201 parenteral dosage form Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000002633 protecting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000004223 radioprotective effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000002511 suppository base Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- HHLJUSLZGFYWKW-UHFFFAOYSA-N triethanolamine hydrochloride Chemical compound Cl.OCCN(CCO)CCO HHLJUSLZGFYWKW-UHFFFAOYSA-N 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
- A61K36/232—Angelica
-
- 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
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
- A61K36/234—Cnidium (snowparsley)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/65—Paeoniaceae (Peony family), e.g. Chinese peony
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/20—Pills, tablets, discs, rods
-
- 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
- A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
Abstract
본 발명은 조혈기능 증진 및 방사선 방호용 생약추출물에 관한 것으로써, 보다 상세하게는 당귀, 천궁, 작약 및 가자(訶子)를 혼합하여 추출한 생약추출물에 관한 것이며 본 발명의 생약추출물은 기존의 생물화학적 면역 및 조혈증강제제들 보다 부작용(side effect)과 독성이 적고 안전성을 보강한 것으로 다양한 질병의 예방 및 회복 또는 노약자를 위한 조혈기능 및 면역기능 증진에 광범위하게 활용될 수 있으며, 방사선 치료시 또는 방사선 피폭시 조혈계를 중심으로 일어나는 방사선 장해의 예방 및 극복에 유용하게 이용될 수 있다.The present invention relates to herbal extracts for enhancing hematopoietic function and radiation protection, and more particularly, to herbal extracts obtained by mixing Angelica, Cheongung, Peony and Gaza (訶子) extracts. It has less side effects, toxicity and safety than chemical immune and hematopoietic agents. It can be widely used for the prevention and recovery of various diseases or for improving hematopoietic and immune function for the elderly. It can be usefully used for the prevention and overcoming of radiation disorders that occur around the hematopoietic system during radiation exposure.
Description
본 발명은 조혈기능 증진 및 방사선 방호용 생약추출물에 관한 것으로써, 보다 상세하게는 당귀, 천궁, 작약 및 가자(訶子)를 혼합하여 추출한 생약추출물에 관한 것이다.The present invention relates to herbal extracts for hematopoietic function enhancement and radiation protection, and more particularly, to herbal extracts obtained by mixing Angelica, Cheongung, Peony and Gaza.
혈액세포는 림프구, 적혈구, 과립구, 대식세포 등과 같은 여러 종류의 세포로 구성되어 있는데, 이러한 세포들은 수명이 한정되어 있으며, 자기복제능력을 가진 공통의 원줄기 세포(stem cells)로부터 계속적으로 만들어져 혈액내로 공급된다. 이러한 혈액 원줄기 세포가 여러 종류의 성숙세포로 증식 분화하는 과정에는 아직도 불확실한 점들이 많으나, 최근에는 혈액 원줄기 세포의 증식과 분화에 관여하는 다양한 조혈인자가 밝혀졌다. 즉, 다양한 단백질인자(사이토카인 등)가 관여할 뿐만 아니라, 혈액 원줄기 세포 주위에 존재하는 간질세포(stromal cells)와의 접촉작용이 매우 중요하다고 생각되고 있다. 조혈조직인 골수(bone marrow) 내에는 내피세포, 지방세포, 섬유아세포, 망상세포, 그리고 대식세포 등이 존재하며 세포 사이에는 파이브로넥틴(fibronectin) 등과 같은 세포외기질(extracellular matrix)이 형성되어 있다.Blood cells are composed of several types of cells, such as lymphocytes, red blood cells, granulocytes, and macrophages. These cells have a limited lifespan and are continuously made from common stem cells with self-replicating ability. Supplied. There are still many uncertainties about the proliferation and differentiation of these blood stem cells into various types of mature cells. Recently, various hematopoietic factors involved in the proliferation and differentiation of blood stem cells have been identified. That is, not only various protein factors (cytokines, etc.) are involved, but also the contact action with stromal cells around blood stem cells is considered to be very important. Endothelial cells, adipocytes, fibroblasts, reticular cells, and macrophages are present in the bone marrow, which is a hematopoietic tissue, and an extracellular matrix such as fibronectin is formed between the cells. .
조혈기능은 골수의 혈액모세포로부터 면역세포를 포함한 여러 가지 혈액세포를 생성하는 기능으로서 면역기능을 통한 생체의 방어력과 밀접한 관계가 있다. 최근 혈액모세포로부터 혈액세포로 분화하는 단계와 각 단계에 작용하는 인자들(cytokines)이 알려지면서 이 인자들을 유전공학적으로 대량생산하여 질병치료에 이용하려는 연구가 활발히 진행되었다. 그러나, 효과를 얻을 수 있을 정도의 대량으로 인체에 투여할 경우 대부분이 심한 독성을 나타내기 때문에 일부 제한적인 용도에만 활용되고 있는 실정이다.Hematopoietic function is a function of generating various blood cells including immune cells from hematopoietic stem cells of bone marrow and is closely related to the defense of the living body through immune function. Recently, as the stages of differentiation from hematopoietic stem cells to blood cells and the factors acting at each stage (cytokines) have been known, studies have been actively conducted to mass-produce these factors and use them for the treatment of diseases. However, when administered to a human body in a large enough amount to be effective, most of them are severely toxic, so they are used only in some limited applications.
면역기능이 여러 가지 질병요인(pathogen)으로부터 생체를 방어한다는 사실은 이미 오래 전에 알려졌는데 이에 따라 질병의 예방 및 치료를 위하여 면역기능을 이용하는 예방접종(vaccination) 및 항독소이용 방법 등이 개발되었으며 최근에는 면역기능의 작용기작이 알려짐에 따라 면역조절물질을 이용하려는 많은 시도가 있었다. 면역조절물질은 비특이적으로 면역세포들을 자극하여 생체의 면역기능을 증진시킴으로써 질병요인에 대한 생체의 방어력을 증강시키는 물질인데, 이와 같은 비특이적 면역조절물질로서 화학합성물질, 미생물 조성물, 생물제제 등을 활용하려는 시도가 계속되어 왔다. 그러나 대부분이 독성 또는 부작용 때문에 실제 적용에는 한계를 보이고 있다.It has been known for a long time that immune function protects living organisms from various pathogens. Therefore, for the prevention and treatment of diseases, vaccination and anti-toxin utilization methods have been developed. As the mechanism of action of immune function is known, many attempts have been made to use immunomodulators. An immunomodulatory substance is a substance that enhances the body's defense against disease factors by stimulating immune cells nonspecifically, thereby enhancing the immune function of the living body. Such nonspecific immunomodulatory substances utilize chemical synthesis materials, microbial compositions, and biologics. Attempts have been made. However, most of them show limitations in practical applications due to toxicity or side effects.
한편, 방사선에 의한 생체 장해의 방호에 있어서도 조혈기능과 면역기능 장해 극복이 중요한 문제로 알려져 있다. 방사선에 의한 장해는 피폭된 선량에 따라 다르게 나타나는데, 초고선량(100∼300 Gy) 피폭 시에는 중추신경계 장해로 인하여 수시간 내지 1∼2일 안에 대부분 사망하게 되고, 고선량(10∼30 Gy) 피폭 시에는 위장관장해(위장관 점막 손상)로 1∼2주안에 많은 수가 사망하게 되며, 중간선량(4∼8 Gy) 피폭 시에는 골수장해로 조혈 및 면역기능 저하를 초래하게 되어 30일 안에 일부가 사망하게 되고, 저선량(1∼2 Gy) 피폭 시에는 저선량장해가 만성적으로 나타나게 된다. 중추신경계 장해 시에는 지금까지 알려진 대응방법이 거의 없으며, 그 이하 선량의 방사선에 의한 장해를 방호하기 위해서는 위장관 장해 억제와 조혈기능 장해 개선이 일차적인 관건이다.On the other hand, overcoming hematopoietic and immune function disorders is also known as an important problem in the protection of biological disorders caused by radiation. Disorders caused by radiation are different depending on the doses exposed, and at high doses (100 to 300 Gy), most of them die within a few hours to 1 to 2 days due to central nervous system disorders, and high doses (10 to 30 Gy). When exposed, gastrointestinal tract obstruction (gastrointestinal mucosa damage) causes a large number of deaths within one to two weeks, and during intermediate doses (4 to 8 Gy), bone marrow disorders result in hematopoietic and immune function declines. Death occurs and chronically low dose disturbances occur at low doses (1-2 Gy). In the case of central nervous system disorders, there are few known countermeasures so far, and in order to protect against the radiation-induced doses of lower doses, suppression of gastrointestinal disorders and improvement of hematopoietic function disorders are the primary issues.
방사선 장해 방호 물질에 대한 연구는 1949년 치올기를 함유한 치올(thiol)복합제가 방사선 방호효과를 갖는다는 보고가 있은 후에, 치올기를 함유한 유기합성물인 WR(Walter Reed; 미육군의학연구소에서 합성한 물질에 붙인 연번의 물질을 총칭함)시리즈의 방호효과에 관한 연구에 집중되었으나 그 독성 때문에 실제 응용에는 한계를 보였다. 1990년 이후에는 미국, 이스라엘, 일본 등에서 디피리다몰(dipyridamole), 아데노신모노포스페이트(adenosine monophosphate), 테트라사클로데카옥사이드(tetracychlorodecaoxide), 아데투론(adeturon), 데옥시스퍼구알린(deoxyspergualin) 등 화학합성제의 효과를 연구하였으나 자체의 심각한 독성 때문에 역시 실제 적용에는 한계를 보였다. 한편으로, 면역반응에 관련된 생체내 인자들과 조혈세포의 분화 및 증식에 관련된 인자들이 밝혀지면서 이들의 일부를 이용하려는 시도가 있었다. 즉, 인터루킨-1(interleukin-1)을 비롯한 면역 자극제, 종양 괴사인자(tumor necrosis factor, TNF), 과립구 집락 형성 촉진인자(granulocyte colony-stimulating factor, GM-CSF)와 같은 조혈세포 증식 유도제 또는 호르몬 등에 의한 방사선 방호 효과를 얻고자 하였으나, 이 역시 독성 때문에 극히 제한적으로 시도되고 있다.The study on radiation-inhibiting substances was carried out in 1949 after the thiol complex containing thiols had a radioprotective effect, and it was synthesized by WR (Walter Reed), an organic compound containing thiols. The study focused on the protective effect of a series of substances attached to a substance. However, due to its toxicity, practical application has been limited. Since 1990, the United States, Israel, Japan and other chemicals such as dipyridamole, adenosine monophosphate, tetratracychlorodecaoxide, adeturon, deoxyspergualin The effectiveness of the synthesis was studied, but due to its serious toxicity, it also showed limitations in practical applications. On the other hand, attempts have been made to utilize some of the in vivo factors involved in immune responses and factors involved in the differentiation and proliferation of hematopoietic cells. That is, hematopoietic proliferation inducers or hormones such as immune stimulants including interleukin-1, tumor necrosis factor (TNF), granulocyte colony-stimulating factor (GM-CSF) In order to obtain a radiation protection effect due to the etc., but this is also very limited attempts because of toxicity.
한편, 최근에는 식품과 생약제제를 중심으로 한 천연물들이 노인성, 퇴행성 질환들에 높은 효능을 보이는 것으로 입증되고 있으며, 이에 따라 이들의 영양생리 및 약리기전에 대한 연구가 활발히 진행되고 있다. 또한, 이들 천연물로부터 생체조절 및 생체방어계를 항진시키는 생리활성물질 탐색이 활발히 진행되어 건강보조식품이나 치료제로서의 실용화 단계에 이르고 있다. 방사선에 의한 조혈기능 장해개선을 위한 천연물의 효능에 관한 연구로는 1988년 프랑스에서 가시오가피의 효능을 검색한 보고 외에, 주로 일본, 대만, 중국을 중심으로 인삼, 천연안료, 만삼, 천궁, 영지와 몇 가지 기존의 한방제에 대한 연구가 최근까지도 진행되고 있으며, 면역조절물질에 대한 연구는 일부 천연물로부터 추출한 유효성분 또는 기존의 한방제의 효과 검증을 중심으로 수행되어 왔다.On the other hand, recently, natural products based on foods and herbal preparations have been shown to show high efficacy against senile and degenerative diseases, and thus, researches on their nutritional physiology and pharmacological mechanisms are being actively conducted. In addition, the search for physiologically active substances that promote bioregulation and defense system from these natural products has been actively progressed, and the practical use as a health supplement food or therapeutic agent has been reached. The research on the efficacy of natural products for the improvement of hematopoietic disorders by radiation, in addition to a report on the efficacy of prickly pear skin in France in 1988, mainly ginseng, natural pigments, ginseng, cheongung, ganoderma Some studies on existing herbal medicines have been conducted until recently, and studies on immunomodulators have been carried out mainly on validating the effects of active ingredients extracted from some natural products or conventional herbal medicines.
이에 본 발명자들은 방사선에 의한 장해가 면역기능 장해와 조혈기능 장해 및 재생조직의 원줄기 세포 손상 등이 복합적으로 일어나는 현상임을 감안하여 각각에 유효한 생약재를 검색하여 당귀, 천궁 및 작약을 동일 무게 비율로 혼합하고, 열탕 추출하여 기존의 면역증강제제들 보다 높은 안정성을 갖는 생약추출물을 제조하여 특허출원한 바 있다(대한민국 특허출원 제 2000-23772호). 그러나, 상기의 발명에서는 면역기능 및 조혈기능에서 광범위하게 효과를 보이긴 하지만 증진 효과의 정도가 대조군에 비하여 3배 정도이며, 방사선 방호효과도 대조군에 비해 1.5배 정도로 미약한 효과를 보였다.Accordingly, the present inventors searched for the herbal medicines effective for each of them in consideration of the fact that radiation-induced disorders are a combination of immune disorders, hematopoietic disorders, and stem cell damage of regenerative tissues. In addition, hot water extract was used to prepare a patent for a herbal extract having a higher stability than the existing immune enhancers (Korean Patent Application No. 2000-23772). However, in the above invention, although broadly effective in immune function and hematopoietic function, the degree of enhancement effect is about three times higher than that of the control group, and the radiation protection effect is about 1.5 times lower than that of the control group.
이에, 본 발명자들은 더 효과적인 새로운 생약제를 검색하여 당귀, 천궁 및 작약에 가자(訶子)를 더하여 동일 무게 비율로 혼합한 후 열탕 추출한 생약추출물을 제조하였으며, 상기 생약추출물이 기존의 생물화학적 면역 및 조혈증강제제들보다 안전성이 높고, 현저한 조혈기능 증진 효과를 보이며, 또한 뛰어난 방사선 방호효과를 나타내는 것을 확인하여 다양한 질병의 예방 및 회복 또는 노약자를 위한조혈기능 및 면역기능 증진에 광범위하게 활용될 수 있으며, 방사선에 의한 조혈계 및 재생조직의 원줄기세포 등의 장해를 종합적으로 방호할 수 있음을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors searched for a new effective herbal medicine, and added the Angelica, Cheongung and Peony to Gaza (訶子), and mixed it in the same weight ratio to prepare a herbal extract extracted with boiling water, and the herbal medicine extract is a conventional biochemical immunity and It has been found to be safer than hematopoietic agents, to show remarkable hematopoietic function enhancement effects, and to show excellent radiation protection effect, and thus can be widely used for prevention and recovery of various diseases or for enhancement of hematopoietic and immune functions for the elderly. The present invention has been completed by confirming that it is possible to comprehensively protect against hematopoietic and radiation induced stem cells of radiation.
본 발명의 목적은 조혈기능 및 면역기능 증진에 광범위하게 활용될 수 있으며 방사선 장해의 예방 및 극복에 유용하게 이용될 수 있는 생약추출물을 제공하는 것이다.An object of the present invention is to provide a herbal extract that can be widely used for improving hematopoietic and immune function and can be usefully used for the prevention and overcoming of radiation disorders.
도 1은 골수세포 배양에서 본 발명의 생약추출물의 조혈전구세포 증식 촉진효과를 나타낸 그래프이고, 1 is a graph showing the hematopoietic progenitor cell proliferation promoting effect of the herbal extract of the present invention in bone marrow cell culture,
1 : 대조군, 2 : 본시료(HIM-2),1: control, 2: main sample (HIM-2),
3 : 당귀, 4 : 천궁,3: donkey, 4: archery,
5 : 작약, 6 : 사물탕,5: peony, 6: samtang,
7 : HIM-1 (당귀, 천궁, 작약을 동일무게비율로 혼합),7: HIM-1 (mixing donkey, celestial and peony at equal weight ratio),
8 : 혼합추출물-1(당귀, 천궁, 작약, 오가피),8: mixed extract-1 (Dang Quai, Cheongung, Peony, Ogapi),
9 : 혼합추출물-2(당귀, 천궁, 작약, 어성초)9: Mixed Extract-2 (Danggui, Cheongung, Peony, Eoseongcho)
도 2는 골수 간질세포 배양에서 본 발명의 생약추출물의 세포 증식 촉진 효과를 나타낸 그래프이고, 2 is a graph showing the cell growth promoting effect of the herbal extract of the present invention in bone marrow stromal cell culture,
1 : 대조군, 2 : 본시료(HIM-2),1: control, 2: main sample (HIM-2),
3 : 당귀, 4 : 천궁,3: donkey, 4: archery,
5 : 작약, 6 : 사물탕,5: peony, 6: samtang,
7 : HIM-17: HIM-1
도 3은 방사선을 조사한 생쥐로부터 분리한 골수세포의 증식에 본 발명의 생약추출물이 미치는 영향을 나타낸 그래프이고, 3 is a graph showing the effect of the herbal extract of the present invention on the proliferation of bone marrow cells isolated from the radiation-treated mice,
A : 대조군, B : 4 Gy 조사 생쥐,A: control group, B: 4 Gy irradiation mice,
C : 8 Gy 조사 생쥐C: 8 Gy Irradiated Mice
도 4는 실험용 생쥐에서 본 발명의 생약추출물의 항체 생성 증진 효과를 나타낸 그래프이고, Figure 4 is a graph showing the antibody production enhancement effect of the herbal extract of the present invention in experimental mice,
1 : 생리식염수, 2 : 본시료(HIM-2), 3 : HIM-1,1: saline solution, 2: main sample (HIM-2), 3: HIM-1,
4 : 당귀, 5 : 천궁, 6 : 작약4: donkey, 5: archery, 6: peony
도 5는 실험용 생쥐에서 본 발명의 생약추출물의 세포성 면역반응 증강 효과를 나타낸 그래프이고, 5 is a graph showing the effect of enhancing the cellular immune response of the herbal extract of the present invention in experimental mice,
1 : 동종동계 + 생리식염수, 2 : 동종이계 + 생리식염수,1: allogeneic + saline, 2: allogeneic + saline,
3 : 동종이계 + 본시료(HIM-2), 4 : 동종이계 + HIM-1,3: allogeneic + main sample (HIM-2), 4: allogeneic + HIM-1,
5 : 동종이계 + 당귀, 6 : 동종이계 + 천궁,5: allogeneic + donkey, 6: allogeneic + celestial,
7 : 동종이계 + 작약7: allogeneic + peony
□ : 수컷, ■ : 암컷□: male, ■: female
도 6은 방사선이 조사된 생쥐에서 본 발명의 생약추출물이 면역세포의 재생성에 미치는 영향을 나타낸 그래프이고, 6 is a graph showing the effect of the herbal extract of the present invention on the regeneration of immune cells in the irradiated mice,
△ : 방사선 대조군△: radiation control
■ : 본시료 투여군(HIM-2)■: Main sample administration group (HIM-2)
도 7은 방사선(12 Gy)을 조사한 생쥐에서 본 발명의 생약추출물이 소장 움의생존에 미치는 영향을 나타낸 그래프이고, 7 is a graph showing the effect of the herbal extract of the present invention on the survival of small intestine in mice irradiated with radiation (12 Gy),
1 : 방사선 대조군, 2 : 본시료(HIM-2), 3 : 천궁,1: radiation control, 2: main sample (HIM-2), 3: uterus,
4 : 당귀, 5 : 백작약, 6 : 사물탕,4: donkey, 5: earl, 6: samultang,
7 : 사군자탕, 8 : 십전대보탕, 9 : 보중익기탕,7: Sagunjatang, 8: Szejeondaebotang, 9: Bojungikgitang,
10 : 삼령백출산, 11 : 귀비탕, 12 : HIM-1,10: Samryeongbaekchulsan, 11: Guibi-tang, 12: HIM-1,
13 : 혼합추출물-1(당귀, 천궁, 작약, 오가피),13: mixed extract-1 (Dangui, Cheongung, Peony, Ogapi),
14 : 혼합추출물-2(당귀, 천궁, 작약, 어성초)14: Mixed Extract-2 (Dangui, Cheongung, Peony, Eoseongcho)
도 8은 방사선(6.5 Gy)을 조사한 생쥐에서 본 발명의 생약추출물이 조혈모세포의 생존에 미치는 영향을 비장내 조혈세포 집락 형성 수로 나타낸 그래프이고, 8 is a graph showing the effect of the herbal extract of the present invention on the survival of hematopoietic stem cells in the splenic hematopoietic cell colony formation in mice irradiated with radiation (6.5 Gy),
1 : 방사선 대조군, 2 : 본시료(HIM-2), 3 : 천궁,1: radiation control, 2: main sample (HIM-2), 3: uterus,
4 : 당귀, 5 : 백작약, 6 : 사물탕,4: donkey, 5: earl, 6: samultang,
7 : 사군자탕, 8 : 십전대보탕, 9 : 보중익기탕,7: Sagunjatang, 8: Szejeondaebotang, 9: Bojungikgitang,
10 : 삼령백출산, 11 : 귀비탕, 12 : HIM-1,10: Samryeongbaekchulsan, 11: Guibi-tang, 12: HIM-1,
13 : 혼합추출물-1(당귀, 천궁, 작약, 오가피),13: mixed extract-1 (Dangui, Cheongung, Peony, Ogapi),
14 : 혼합추출물-2(당귀, 천궁, 작약, 어성초)14: Mixed Extract-2 (Dangui, Cheongung, Peony, Eoseongcho)
도 9는 방사선(4 Gy)을 조사한 생쥐에서 본 발명의 생약추출물이 조혈기능 회복에 미치는 영향을 혈액내 재생성된 혈액세포 중 백혈구 수로 나타낸 그래프이고, 9 is a graph showing the effect of the herbal extract of the present invention on the recovery of hematopoietic function in the mice irradiated with radiation (4 Gy) as the number of leukocytes in the blood cells regenerated in the blood,
△ : 방사선 대조군, ■ : 본시료 투여군(HIM-2)△: radiation control group, ■: main sample administration group (HIM-2)
도 10은 방사선(4 Gy)을 조사한 생쥐에서 본 발명의 생약추출물이 조혈기능회복에 미치는 영향을 혈액내 재생성된 혈액세포 중 적혈구 수로 나타낸 그래프이고, 10 is a graph showing the effect of the herbal extract of the present invention on hematopoietic function recovery in the mice irradiated with radiation (4 Gy) as the number of red blood cells in the blood cells regenerated in the blood,
△ : 방사선 대조군, ■ : 본시료 투여군(HIM-2)△: radiation control group, ■: main sample administration group (HIM-2)
도 11은 방사선(2 Gy)을 조사한 생쥐에서 본 발명의 생약추출물이 소장 움 세포의 자사(apoptosis)에 미치는 영향(억제효과)을 나타낸 그래프이다. 11 is a graph showing the effect (inhibitory effect) of the herbal extract of the present invention on small cell intestinal cells (apoptosis) in the radiation (2 Gy) irradiated mice.
1 : 방사선 대조군, 2 : 본시료(HIM-2), 3 : 천궁,1: radiation control, 2: main sample (HIM-2), 3: uterus,
4 : 당귀, 5 : 백작약, 6 : 사물탕,4: donkey, 5: earl, 6: samultang,
7 : 사군자탕, 8 : 십전대보탕, 9 : 보중익기탕,7: Sagunjatang, 8: Szejeondaebotang, 9: Bojungikgitang,
10 : 삼령백출산, 11 : 귀비탕, 12 : HIM-1,10: Samryeongbaekchulsan, 11: Guibi-tang, 12: HIM-1,
13 : 혼합추출물-1(당귀, 천궁, 작약, 오가피),13: mixed extract-1 (Dangui, Cheongung, Peony, Ogapi),
14 : 혼합추출물-2(당귀, 천궁, 작약, 어성초)14: Mixed Extract-2 (Dangui, Cheongung, Peony, Eoseongcho)
상기 목적을 달성하기 위하여, 본 발명은 조혈기능과 면역기능 증진 및 방사선 방호용 생약추출물로서 당귀, 천궁, 작약 및 가자(訶子)를 혼합하여 열탕 추출한 생약추출물을 제공한다.In order to achieve the above object, the present invention provides a herbal extract extracted by boiling water mixed with Angelica, Cheongung, Peony and Gaza (생 子) as a herbal extract for hematopoietic function and immune function enhancement and radiation protection.
또한, 본 발명은 상기 생약추출물을 유효성분으로 포함하는 약학적 조성물 및 기능성 식품을 제공한다.In addition, the present invention provides a pharmaceutical composition and a functional food comprising the herbal extract as an active ingredient.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 조혈기능과 면역기능 증진 및 방사선 방호용 생약추출물로서 당귀, 천궁, 작약 및 가자(訶子)를 혼합하여 열탕 추출한 생약추출물을 제공한다.The present invention provides a herbal extract extracted by boiling hot water mixed with Angelica, Cheongung, Peony and Gaza (생 子) as a herbal extract for hematopoietic function and immune function enhancement and radiation protection.
구체적으로 본 발명에서는 기존의 수종 한방제들 및 그 구성 생약재와 기존의 처방들을 변형한 제제들을 대상으로 조혈기능과 면역기능 증진 및 방사선 방호 효과를 비교 검색하였다. 그 결과를 바탕으로 조혈기능 및 면역기능 증진과 동시에 방사선 방호 효과의 더 높은 활성을 나타내는 당귀, 천궁, 작약 및 가자(訶子)로 구성된 혼합 생약추출물을 제조하였다.Specifically, the present invention was compared and searched for the effect of hematopoietic function, immune function enhancement and radiological protection against the existing herbal medicines and its constituent herbal medicines and formulations modified from existing prescriptions. Based on the results, a mixed herbal extract consisting of Angelica, Cheongung, Peony and Gaza exhibited higher activity of hematopoietic and immune function and at the same time higher activity of radiation protection.
본 발명의 생약추출물은 식품소재로 이용할 수 있도록 고시되어 있는 생약재의 3 가지 즉 당귀, 천궁 및 작약 그리고 생약재로서 독성이 비교적 적은 가자(訶子)를 사용하였으며, 상기 4 가지 생약재를 혼합하고 열탕 추출하여 혼합조성물로 제조되었다.The herbal extracts of the present invention used three kinds of herbal medicines that have been announced to be used as food materials, namely, Angelica, Cheongung and Peony, and Gaza (訶子) having relatively low toxicity as herbal medicines, and mixing the four herbal medicines and extracting boiling water. To prepare a mixed composition.
본 발명의 생약추출물의 제조방법은The preparation method of the herbal extract of the present invention
1) 당귀, 천궁, 작약 및 가자의 혼합 생약재에 물을 가하는 단계,1) adding water to the mixed herbal medicine of Angelica, Cheongung, Peony and Gaza,
2) 단계 1의 물을 가한 생약재를 가열하여 추출하는 단계; 및2) heating and extracting the herbal medicine to which the water of step 1 is added; And
3) 단계 2에서 추출한 생약재를 감압 농축기로 농축하는 단계로 구성된다.3) Concentrating the herbal medicines extracted in step 2 with a vacuum concentrator.
상기 단계 1에서 물은 총 생약재 무게의 5내지 20배 가하는 것이 바람직하며, 총 생약재 무게의 10배를 가하는 것이 더욱 바람직하다. 또한, 당귀, 천궁, 작약 및 가자는 10-35 : 10-35 : 10-35 : 10-35 중량부의 비율로 혼합하는 것이 바람직하며 각각 동일한 중량부로 혼합하는 것이 더욱 바람직하다.In step 1, the water is preferably added 5 to 20 times the total weight of the herbal medicines, more preferably 10 times the total weight of the herbal medicines. In addition, Angelica, Cheongung, Peony and Gaza are preferably mixed in the ratio of 10-35: 10-35: 10-35: 10-35 parts by weight, and more preferably in the same parts by weight.
상기 단계 2에서, 추출은 3시간씩 3회 가열하여 추출하는 것이 바람직하다. 단계 3에서 농축한 생약재는 동결 건조시켜 분말로 제조하거나 그 자체로 이용할 수 있다.In step 2, the extraction is preferably extracted by heating three times for 3 hours. The herbal medicines concentrated in step 3 may be lyophilized to prepare a powder or used as such.
본 발명의 생약추출물은 조혈기능 증진 효과를 나타낸다. 조혈조직인골수(bone marrow) 내에는 내피세포, 지방세포, 섬유아세포, 망상세포, 그리고 대식세포 등이 존재하며 세포 사이에는 파이브로넥틴(fibronectin) 등과 같은 세포외기질(extracellular matrix)이 형성되어 있다. 이러한 세포들과 세포외기질 등이 혈구의 분화 증식에 적합한 조혈미세환경을 형성하는 것으로 알려져 있다. 따라서, 본 발명자들은 조혈미세환경을 조절할 수 있는 물질이 조혈기능을 조절할 수 있을 것으로 생각하고, 실제적으로 본 발명의 생약추출물이 시험관 내에서 골수 간질세포를 증식시킬 뿐만 아니라, 조혈 전구세포의 분화 및 증식을 촉진시킨다는 것을 확인하여, 본 발명의 생약추출물이 조혈 미세환경에서의 조혈계의 기능증진을 유도함을 밝혔다.Herbal extract of the present invention shows an effect of enhancing hematopoietic function. In the bone marrow, hematopoietic tissue, endothelial cells, adipocytes, fibroblasts, reticular cells, and macrophages are present. Extracellular matrix such as fibronectin is formed between the cells. . These cells and extracellular matrix are known to form a hematopoietic microenvironment suitable for differentiation and proliferation of blood cells. Therefore, the present inventors believe that a substance capable of regulating the hematopoietic microenvironment can regulate hematopoietic function, and in fact, the herbal extract of the present invention not only proliferates bone marrow stromal cells in vitro, but also differentiates hematopoietic progenitor cells and Confirming that it promotes proliferation, the herbal extract of the present invention was found to induce the enhancement of the hematopoietic system in the hematopoietic microenvironment.
본 발명의 생약추출물은 정상 생쥐에 항원으로서 면양적혈구(sheep red blood cell, SRBC)를 주사한 경우 항체생산 세포수를 증가시키며, 동종이계(同種異系, allogeneic) 면역세포를 이식하였을 때의 반응(GraftversusHost Reaction), 즉 T-세포매개 세포성 면역반응을 증가시키는데, 이는 상기 생약추출물이 면역기능 증진 효과를 나타냄을 의미한다.The herbal extract of the present invention increases the number of antibody-producing cells when injecting sheep red blood cells (SRBC) as antigens into normal mice, and responds to transplantation of allogeneic immune cells. (Graft versus Host Reaction), ie T-cell mediated cellular immune response is increased, which means that the herbal extracts exhibit an immune function enhancing effect.
상기의 조혈기능 증진효과는 방사선 조사된 생쥐에서도 효과적으로 나타나는데, 이는 본 발명의 생약추출물이 방사선 방호에도 효과적임을 의미한다. 본 발명자들은 하기와 같은 사실에 근거하여 본 발명에서 제공하는 생약추출물의 방사선 방호효과를 구체적으로 확인하였다.The hematopoietic function-promoting effect is also effective in irradiated mice, which means that the herbal extract of the present invention is also effective in radiation protection. The present inventors specifically confirmed the radiation protection effect of the herbal extract provided by the present invention based on the following facts.
생체를 구성하고 있는 조직 또는 세포에는 방사선에 피폭되는 경우 장해를 받기 쉬운 방사선에 민감한 조직과 상대적으로 저항성을 보이는 조직이 있다. 방사선 장해 방호는 위장관장해를 나타내는 선량 이하의 방사선에 피폭된 경우를 주 대상으로 하고 있다. 방사선에 의한 장해를 방호함에 있어서 위장관장해 억제와 골수장해로 인한 조혈 및 면역기능 저하의 극복이 관건이다. 본 발명자들은 상기 생약추출물을 생쥐에 투여하고 위장관장해 억제와 조혈기능 및 면역기능 장해개선 여부를 확인하였다. 본 발명의 생약추출물은 고선량의 방사선을 조사한 경우 소장의 움(intestinal crypt)을 방사선으로부터 방호하여 생존 움의 수를 증가시키고, 중간 선량의 방사선을 조사한 생쥐에서 조혈모세포를 방호하여 혈액세포와 면역세포의 재생성을 촉진시키며, 저선량의 방사선을 조사한 생쥐에서는 소장 움 세포의 사멸(apoptosis) 정도를 억제하는 효과를 나타내었다. 상기 결과는 본 발명의 생약추출물이 방사선 방호에 효과적임을 의미한다.Tissue or cells constituting the living body include tissues that are relatively resistant to radiation-sensitive tissues that are susceptible to radiation exposure. Radiation obstruction protection is primarily intended for exposure to radiation below doses that indicate gastrointestinal disorders. In the prevention of radiation-induced disorders, the prevention of gastrointestinal disorders and the overcoming of hematopoietic and immune function deterioration due to bone marrow disorders are key. The present inventors administered the herbal extract to mice and confirmed the inhibition of gastrointestinal disorders and the improvement of hematopoietic and immune function disorders. The herbal extract of the present invention increases the number of viable worms by protecting the intestinal crypt from radiation when irradiated with high doses of radiation, and protects the hematopoietic stem cells from mice with medium doses of radiation to protect blood cells and immunity. Increasing the regeneration of cells and low-irradiation mice showed the effect of inhibiting the degree of apoptosis of small intestinal cells. The results indicate that the herbal extracts of the present invention are effective for radiation protection.
또한, 본 발명은 상기 생약추출물을 유효성분으로 함유하는 약학적 조성물 및 기능성 식품을 제공한다.The present invention also provides a pharmaceutical composition and functional food containing the herbal extract as an active ingredient.
본 발명의 생약추출물은 이를 유효성분으로 포함하는 약학적 조성물로서 여러 가지 질병의 예방 및 회복 또는 노약자를 위한 조혈 및 면역 기능 증진에 광범위하게 활용될 수 있으며, 방사선 치료시 또는 방사선 피폭에 따른 방사선 장해 시 조혈계 장해의 극복 및 재생조직 원줄기세포 장해의 극복 등에 활용될 수 있다. 또한 상기 생약재 조합의 혼합비율을 최종 제품의 상품성과 소비자 기호도에 따라서 변형시키거나, 다른 식품소재를 첨가하여 기능성 식품으로 제조할 수 있다.The herbal extract of the present invention is a pharmaceutical composition comprising the same as an active ingredient can be widely used for the prevention and recovery of various diseases or to improve the hematopoietic and immune functions for the elderly, radiation interference during radiation therapy or radiation exposure It can be used for overcoming hematopoietic disorder and regenerative tissue stem cell disorder. In addition, the mixing ratio of the herbal medicine combination may be modified according to the commerciality and consumer preference of the final product, or may be prepared as a functional food by adding other food materials.
본 발명의 생약추출물을 유효성분으로 포함하는 약학적 조성물은 임상투여시에 경구 또는 비경구로 투여가 가능하며, 일반적인 의약품제제의 형태로 사용될 수 있다.The pharmaceutical composition comprising the herbal extract of the present invention as an active ingredient may be administered orally or parenterally during clinical administration, and may be used in the form of a general pharmaceutical formulation.
즉, 본 발명의 약학적 조성물은 실제 임상투여시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 생약추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(Calcium carbonate), 수크로스(Sucrose) 또는 락토오스(Lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용 될 수 있다.In other words, the pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used Or using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. Such solid preparations include at least one excipient such as starch, calcium carbonate, water, and the like in the herbal extract of the present invention. It is prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
본 발명의 생약추출물의 유효용량은 5 내지 300 mg/kg 이고, 바람직하기로는 10 내지 150 mg/kg 이며, 하루 1 내지 3회 투여할 수 있다.The effective dose of the herbal extract of the present invention is 5 to 300 mg / kg, preferably 10 to 150 mg / kg, may be administered 1 to 3 times a day.
상기 생약추출물은 생쥐를 대상으로 한 급성독성검사 결과 경구 투여 시에는 2 g/kg까지 독성변화를 전혀 나타내지 않았으며, 복강내 주사시 50% 치사량(LD50)이 0.4 g/kg 이상으로 나타나 독성이 경미한 물질로 판단되었다.The herbal extracts showed no change in toxicity up to 2 g / kg when administered orally as a result of an acute toxicity test in mice, and 50% lethal dose (LD 50 ) was 0.4 g / kg or more when intraperitoneally injected. It was judged to be a slight substance.
이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
<실시예 1> 당귀, 천궁, 작약 및 가자(訶子)를 포함한 생약추출물의 제조<Example 1> Preparation of herbal extracts, including Angelica, Cheongung, Peony and Gaza
당귀, 천궁, 작약 및 가자(訶子)의 생약재 4가지를 음건하고 세절하여 동일한 무게 비율로 혼합한 후 총 무게의 10배 량의 증류수를 가하여 3 시간씩 3 회 약탕기에서 끓여 추출하였다. 상기 추출물을 여과한 후 감압 농축기로 농축한 다음 동결건조하여 본 발명의 생약추출물을 제조하였다. 실험에 사용하기 위하여 증류수에 10~100 mg/ml의 농도로 녹이고 무균 여과지(Millipore membrane, 0.45 ㎛)로 여과하여 무균 상태를 유지하였다.Four herbal medicines of Angelica, Cheonggung, Peony and Gaza (訶子) were shaded, shredded and mixed at the same weight ratio, and then distilled water of 10 times the total weight was added and boiled in a medicine bath three times for 3 hours. The extract was filtered and concentrated under reduced pressure concentrator and then lyophilized to prepare the herbal extract of the present invention. For use in the experiment was dissolved in distilled water at a concentration of 10 ~ 100 mg / ml and filtered with a sterile filter paper (Millipore membrane, 0.45 ㎛) to maintain a sterile state.
<실험예 1> 조혈기능 증강효과 측정Experimental Example 1 Measurement of Hematopoietic Function Enhancing Effect
<1-1> 조혈전구세포(골수간세포, bone marrow stem cells)의 증식효과<1-1> Proliferative effect of hematopoietic progenitor cells (bone marrow stem cells)
골수세포 배양계에 본 발명의 생약추출물을 첨가하여 본 발명의 생약추출물이 조혈전구세포의 증식에 미치는 효과를 확인하였다. 골수세포는 생쥐의 대퇴골로부터 분리하여 사용하였다.The herbal extract of the present invention was added to the bone marrow cell culture system to confirm the effect of the herbal extract of the present invention on proliferation of hematopoietic progenitor cells. Bone marrow cells were used from the femur of mice.
실험에 사용한 생쥐(C57BL/6 mouse)는 실험에 사용하기 전까지 온도가 22 ± 2℃, 습도가 55 ~ 60%로 유지되며 명암이 12시간씩 조절되는 표준사육 환경에서 생육하였고 고형사료와 물을 제한 없이 공급하였다.The mice used in the experiment (C57BL / 6 mouse) were grown in a standard breeding environment in which the temperature was maintained at 22 ± 2 ° C, the humidity was 55 to 60%, and the contrast was adjusted for 12 hours before being used in the experiment. Supply without limitation.
골수세포를 얻기 위하여, 생쥐를 경추 탈구법으로 희생시킨 뒤 대퇴골을 무균적으로 적출한 다음 주사기를 이용하여 대퇴골 내부의 골수를 씻어내어 골수세포를 분리하였다. 골수세포를 장기간 배양하면, 배양접시에 부착하는 간질세포(stromal cells)와 부착하지 않고 배양액 내로 분화 증식되어 나오는 비부착성 세포(nonadherent cells)를 관찰할 수 있는데, 간질세포와의 접촉작용을 통해 증식하는 비부착성 세포는 단구/대식세포, 혈소판, 적혈구 등으로 분화가 가능한 조혈전구세포로서 이들의 증식에 본 발명의 생약추출물이 미치는 효과를 확인하였다. 이러한 세포들을 배양 상층액과 같이 회수하여 혈구계수판으로 비부착성 세포수를 계수하였다.In order to obtain the bone marrow cells, mice were sacrificed by cervical dislocation, and the femur was removed aseptically, and the bone marrow inside the femur was washed using a syringe to separate the bone marrow cells. After prolonged culturing of bone marrow cells, nonadherent cells that differentiate and proliferate into culture medium without adherence to stromal cells that adhere to the culture plate can be observed. Non-adherent cells to proliferate are hematopoietic progenitor cells capable of differentiation into monocytes / macrophages, platelets, erythrocytes, etc., and confirmed the effect of the herbal extract of the present invention on their proliferation. These cells were harvested together with the culture supernatant and the number of nonadherent cells was counted with a hemocytometer.
그 결과, 골수세포 배양계에 본 추출물을 첨가하였을 때 분화 증식되는 비부착성의 조혈전구세포가 대조군보다 훨씬 많이 증식되는 것을 확인하였다 (도 1).As a result, it was confirmed that the non-adherent hematopoietic progenitor cells that differentiated and proliferated when the present extract was added to the bone marrow cell culture system proliferated much more than the control group ( FIG. 1 ).
<1-2> 골수 간질세포(Bone marrow stromal cells)의 증식 효과<1-2> Proliferative effect of bone marrow stromal cells
골수간질세포 배양계에 본 발명의 생약추출물을 첨가하여 증식에 미치는 효과를 관찰하였다. 먼저 골수세포를 실험예 <1-1>과 같은 방법으로 생쥐 대퇴골로부터 분리하여 24시간 동안 배양한 후, 배양액 내에 부유하는 조혈전구세포를 배양액과 함께 제거하여 배양접시에 부착하는 간질세포만을 분리하여 준비하였다. 분리된 골수 간질세포를 장기간 배양하면서, 일주일에 한번씩 배지의 1/2을 새로운 배지로 교환하고, 생약추출물을 첨가하여 2 ~ 3주일 정도 계속 배양한 후 증식한 간질세포 수를 세어서 증식 정도를 측정하였다.The effect of the herbal extract of the present invention on the proliferation was observed in the myeloid stromal cell culture system. First, the bone marrow cells were isolated from the mouse femur in the same manner as in Experimental Example <1-1>, and cultured for 24 hours. Then, the hematopoietic progenitor cells suspended in the culture medium were removed together with the culture medium to separate only the stromal cells attached to the culture dish. Ready. While culturing isolated bone marrow stromal cells for a long time, exchange 1/2 of the medium with fresh medium once a week, continue the culture for 2 to 3 weeks with the addition of herbal extracts, and count the number of proliferated stromal cells. Measured.
그 결과, 본 추출물에 의한 간질세포의 현저한 증식 효과를 확인할 수 있었다 (도 2).As a result, the proliferative effect of the stromal cells by the extract was confirmed ( Fig. 2 ).
상기 결과는 본 발명의 생약추출물이 조혈전구세포의 증식뿐만 아니라 조혈 미세환경 내의 보조세포인 간질세포의 증식도 촉진시킴으로써 조혈계의 기능증진을 유도함을 의미한다.The results indicate that the herbal extract of the present invention induces the enhancement of the hematopoietic system by promoting the proliferation of hematopoietic progenitor cells as well as the proliferation of stromal cells, which are auxiliary cells in the hematopoietic microenvironment.
<1-3> 방사선 조사에 대한 골수세포 회복 효과<1-3> Effect of Bone Marrow Cell Recovery on Irradiation
상기 생약추출물이 방사선 조사로 인한 골수세포 장해를 회복시킬 수 있는지를 알아보기 위하여, 감마선 조사를 한 생쥐의 골수세포를 분리한 후 본 발명의 생약추출물을 첨가한 배지에서 일정기간 배양한 다음 회복되는 정도를 세포수로 비교하였다.In order to find out whether the herbal extract can recover the bone marrow cell damage caused by irradiation, after separating the bone marrow cells of the gamma-irradiated mice and incubated in the medium containing the herbal extract of the present invention for a certain period of time The extent was compared by cell number.
그 결과, 감마선 조사를 하지 않은 대조군에서도 생약추출물의 첨가에 의해 약 1.5배 정도로 세포수가 증가되었다. 50% 치사량의 감마선인 8 Gy을 조사한 생쥐에서 분리한 골수세포는 그 생존정도가 약 7% 정도밖에 되지 않았으나, 본 발명의 생약추출물 첨가에 의해 약 1.7배 정도로 생존 세포수가 증가되었다. 또한, 4 Gy의 감마선을 조사한 생쥐에서 분리한 골수세포는 약 10% 정도밖에 생존하지 않았으나, 본 발명의 생약추출물 첨가에 의해 약 2배 정도로 생존 세포수가 증가됨을 확인하였다(도 3). 상기 결과는 본 발명의 생약추출물이 방사선에 피폭된 생쥐에서 골수세포의 회복을 유도함을 의미한다.As a result, the number of cells increased about 1.5-fold by the addition of the herbal extract even in the control group without gamma irradiation. Bone marrow cells isolated from mice irradiated with 50% lethal gamma-ray 8 Gy survived only about 7%, but the number of viable cells increased by about 1.7 times by the addition of the herbal extract of the present invention. In addition, the bone marrow cells isolated from mice irradiated with 4 Gy gamma-rays survived only about 10%, but it was confirmed that the number of viable cells increased by about two times by the addition of the herbal extract of the present invention ( FIG. 3 ). The results indicate that the herbal extract of the present invention induces the recovery of bone marrow cells in the mice exposed to radiation.
<실험예 2> 면역기능 증강효과Experimental Example 2 Immune Function Enhancing Effect
<2-1> 생체내의 항체생성 증강 효과<2-1> Enhancement effect of antibody production in vivo
본 발명의 생약추출물이 생체내 면역기능 중 체액성 면역반응인 항체 생성능에 미치는 효과를 알아보기 위하여, 생쥐에 추출물을 투여하고 항원으로 면양적혈구(Sheep Red Blood Cell, SRBC)를 주사한 후 초기 면역반응에서 면양적혈구에 대한 항체를 생성하는 세포 수를 측정하였다.In order to determine the effect of the herbal extract of the present invention on the ability to produce a humoral immune response of the immune function in vivo, the initial immunization after administration of the extract to the mouse and injection of sheep red blood cells (SRBC) with antigen The number of cells producing antibodies to sheep blood cells in the reaction was measured.
실험에 사용한 생쥐(C57BL/6 mouse)는 실험예 <1-1>과 동일한 표준 사육조건에서 사육하였다.The mice used for the experiment (C57BL / 6 mouse) It was bred under the same standard breeding conditions as in Experimental Example <1-1>.
항체생성 세포수 측정(Hemolytic plaque forming cell assay)을 위하여, 먼저 본 발명의 생약추출물의 최적용량을 정하여 생쥐 마리당(25 g) 0.25 ㎖씩 복강내 주사하였다. 다음날, 면양적혈구(SRBC)를 생쥐 마리당 2 ×108개씩 정맥주사한 후 2일간 계속해서 본 발명의 생약추출물을 복강내 주사하였다. 면양적혈구 주사 후 3 일째에 마우스를 경추탈골로 희생시키고 무균적으로 비장세포를 분리한 후,비장세포(7 ×105세포/0.1 ㎖), 20% 면양적혈구(SRBC) 0.1 ㎖ 및 0.5% 아가로즈(agarose) 1.6 ㎖을 시험관에서 혼합하여 배양접시(dish)에 부은(pour)후 2시간 동안 세포배양기(CO2-incubator)에서 배양하였다. 이후 보체(GPC,1/70)를 첨가하여 2시간 더 배양한 후 항체를 생산하는 림프구 주변에 형성된 용혈 플라크(plaque)를 계수하고 희석 배수를 고려하여 비장세포 전체 중 항체를 생산하는 세포수(plaque forming cells/spleen, PFC/spleen)를 산출하였다.In order to measure the antibody-producing cell number (Hemolytic plaque forming cell assay), firstly, the optimal dose of the herbal extract of the present invention was determined and injected intraperitoneally with 0.25 ml of mice per head (25 g). On the following day, intravenous injection of 2 × 10 8 rat sheep blood cells (SRBC) per mouse was continued intraperitoneally for 2 days. Three days after inoculation, the mice were sacrificed with cervical distal bone and splenocytes isolated aseptically, followed by splenocytes (7 × 10 5 cells / 0.1 ml), 20 ml of SBC (0.1 ml) and 0.5% agar. 1.6 ml of rose (agarose) was mixed in a test tube and poured into a culture dish (pour) and incubated in a cell incubator (CO 2 -incubator) for 2 hours. After 2 hours of incubation with the addition of complement (GPC, 1/70), the hemolytic plaques formed around the lymphocytes producing the antibody were counted and the number of cells producing the antibody in the whole splenocytes in consideration of the dilution factor ( plaque forming cells / spleen, PFC / spleen).
그 결과, 생약추출물의 투여에 의하여 항체생성 세포 수가 현저히 증가함을 확인하였다(도 4).As a result, it was confirmed that the number of antibody-producing cells was significantly increased by administration of the herbal extract ( FIG. 4 ).
<2-2> 생체내 세포성 면역반응의 증강효과<2-2> Enhancement effect of cellular immune response in vivo
생체내 면역기능 중 세포성 면역반응에 미치는 본 발명의 생약추출물의 효과를 확인하기 위하여, 생쥐에 동종이계(同種異系, allogeneic)의 면역세포(immune cells)를 이식하여 이식된 면역세포에 의한 세포성 면역반응(Graft verus Host Reaction, GVH)을 유발시킨 후 본 발명의 생약추출물을 투여하였다.In order to confirm the effect of the herbal extract of the present invention on the cellular immune response during in vivo immune function, the immune cells transplanted by transplanting allogeneic immune cells into mice After inducing cellular immune response (Graft verus Host Reaction, GVH), the herbal extract of the present invention was administered.
실험에 사용한 생쥐 2종은 C57BL/6와 DBA/2이었으며, 잡종 생쥐인 BDF1(C57BL/6×DBA/2)은 C57BL/6 암컷과 DBA/2 수컷을 교배시켜 얻었다.The two mice used in the experiment were C57BL / 6 and DBA / 2. Hybrid mice, BDF1 (C57BL / 6 × DBA / 2), were obtained by crossing C57BL / 6 females and DBA / 2 males.
세포성 면역반응(Graftvs.Host Reaction, GVH)을 측정하기 위하여, 먼저BDF1(C57BL/6×DBA/2) 마우스를 수용체로 하였으며, 동종이계(同種異系, allogeneic)의 이식 세포(graft)로는 C57BL/6 생쥐의 비장림프구를 사용하였다.비장세포를 생쥐 마리당 1×107개(0.2 ㎖)씩 꼬리정맥으로 주사하여 이식하였으며, 생약추출물의 투여는 적정용량이 되도록 조정하여 이식시키는 날로부터 7 일간 복강주사(0.2 ㎖/마리)로 실시하였다. 7일 후 비장을 적출하여 무게를 재고 체중으로 나누어 하기 수학식 1에 의한 비장지표(spleen index)를 산출하였다.In order to measure the cellular immune response (Graft vs. Host Reaction, GVH), BDF1 (C57BL / 6 × DBA / 2) mice were first used as receptors, and allogeneic grafts were transplanted. The spleen lymphocytes of C57BL / 6 mice were used. The splenocytes were implanted into the tail vein by 1 × 10 7 mice (0.2 ml) per mouse, and the administration of the herbal extracts was adjusted to an appropriate dose from the day of transplantation. It was carried out by intraperitoneal injection (0.2 ml / horse) for 7 days. After 7 days, the spleen was extracted, and the weight was divided by weight to calculate a spleen index according to Equation 1 below.
산출된 비장지표의 분석결과, 세포성 면역반응(GVH)이 생약추출물의 투여에 의하여 현저하게 증폭되는 것을 확인하였다(도 5).As a result of analysis of the calculated spleen indicator, it was confirmed that cellular immune response (GVH) is significantly amplified by administration of the herbal extract ( FIG. 5 ).
<3-3> 방사선이 조사된 생쥐에서 비장내 면역세포의 재생성 촉진효과<3-3> Promoting Regeneration of Immune Cells in the Spleen in Irradiated Mice
본 발명의 생약추출물이 방사선이 조사된 생쥐에서 면역세포의 재생성에 미치는 효과를 확인하기 위하여 하기와 같은 실험을 수행하였다. 시료 투여는 생쥐에 방사선을 조사하기 전 36시간과 12시간에 생쥐 마리 당 1 mg씩 복강내로 주사하였다. 방사선 조사 후 시일 경과에 따라 비장을 적출하고 비장내 면역세포를 분리하여 계수하였다.In order to confirm the effect of the herbal extract of the present invention on the regeneration of immune cells in the irradiated mice, the following experiment was performed. Sample administration was intraperitoneally injected at 1 mg per mouse at 36 and 12 hours prior to irradiation. After irradiation, the spleens were removed and the immune cells in the spleens were separated and counted.
비장림프구를 얻기 위하여, 생쥐를 경추 탈구법으로 희생시킨 뒤 복부를 절개하여 비장을 무균적으로 적출하고 멸균된 핀셋과 수술용 칼로 지분거려 생리식염수(HBSS)에 세포를 부유시켰다. 상기 비장세포 부유액을 2분간 정치시켜 부유되지않은 세포 덩어리를 제거한 후, 트리스-염화암모늄 완충용액(Tris-NH4Cl용액)을 가하여 적혈구를 제거하고 HBSS로 2회 세척하여 비장 림프구를 얻었다. 이를 트리판블루(Trypan blue) 염색법을 이용하여 세포수를 측정하였다.To obtain splenic lymphocytes, mice were sacrificed by cervical dislocation and the abdomen was excised to remove the spleen aseptically, and the cells were suspended in physiological saline (HBSS) by sterilization with sterile tweezers and a surgical knife. The splenocyte suspension was allowed to stand for 2 minutes to remove unsuspended cell mass, and then tris-ammonium chloride buffer solution (Tris-NH 4 Cl solution) was added to remove red blood cells and washed twice with HBSS to obtain spleen lymphocytes. The cell number was measured using Trypan blue staining.
그 결과, 생쥐 비장내 면역세포 수는 방사선 조사 후 급격히 감소하였으며, 그 후 서서히 회복되었으나, 본 발명의 생약추출물 투여군에서는 방사선 조사 후 3 주째부터 대조군에 비해 현저하게 면역세포의 수가 증가되었다(도 6). 이는 본 발명의 생약추출물이 방사선에 피폭된 생쥐에서 면역세포의 재생성을 촉진함을 의미한다.As a result, the number of immune cells in the mouse spleen decreased sharply after irradiation, and then recovered slowly, but the number of immune cells in the herbal extract administration group of the present invention increased significantly compared to the control group from 3 weeks after irradiation ( Fig. 6). ). This means that the herbal extract of the present invention promotes the regeneration of immune cells in mice exposed to radiation.
<실험예 3> 방사선 방호효과Experimental Example 3 Radiation Protection Effect
<3-1> 위장관장해 방호효과<3-1> Protective effect of gastrointestinal disorders
본 발명의 생약추출물이 고선량 방사선에 의한 위장관장해를 방호할 수 있는지를 확인하기 위하여 소장 움 생존 증가 효과(Intestinal crypt survival)을 관찰하였다.Intestinal crypt survival was observed to determine whether the herbal extracts of the present invention could protect gastrointestinal disorders caused by high dose radiation.
먼저, 소장 움 생존 증가 효과를 확인하기 위하여 생쥐를 방사선에 피폭시키기 전에 생약추출물을 주사한 다음 12 Gy의 방사선에 노출시켰다. 상기 생약추출물의 투여는 생쥐 마리 당 1 ㎎의 용량으로 방사선 조사 전 36 및 12시간 전에 복강 내로 2회 주사하였다. 그 후 3.5일째에 생쥐를 희생시켜 소장부위를 채취하고, 각 생쥐 당 8 내지 10개의 소장편을 통상적인 방법에 따라 파라핀 포매하여 절편을제작하였다. 각 생쥐 당 8개의 종절된 소장표본의 가장자리에 위치하는 소장 움을 광학현미경으로 관찰하고 그 수를 계수하였다.First, the herbal extracts were injected and then exposed to 12 Gy of radiation before the mice were exposed to radiation to confirm the effect of increasing small bowel survival. The herbal extract was injected twice intraperitoneally 36 and 12 hours before irradiation at a dose of 1 mg per mouse. Thereafter, the small intestine was sacrificed at 3.5 days, and small intestine sections were collected, and sections were prepared by embedding paraffin in 8 to 10 small intestines per mouse. The small intestine located at the edge of eight terminated small intestine specimens for each mouse was observed by light microscopy and counted.
그 결과, 생존 소장 움의 수가 정상 대조군에서는 평균 157.3개, 방사선 피폭 대조군에서는 21.3개, 본 생약추출물 투여군에서는 34.4개로 나타나, 본 발명의 생약추출물이 생존 소장 움의 수를 유의하게(p<0.05) 증가시킴을 확인하였다(도 7). 이는 본 발명의 생약추출물이 방사선에 의한 위장관 장해를 방호할 수 있음을 의미한다.As a result, the average number of viable small intestine was 157.3 in the normal control group, 21.3 in the radiation exposure control group and 34.4 in the herbal extract administration group, and the herbal extract of the present invention significantly (p <0.05) Increase was confirmed ( FIG. 7 ). This means that the herbal extract of the present invention can protect the gastrointestinal tract obstruction by radiation.
<3-2> 조혈모세포 방호효과<3-2> Hematopoietic stem cell protective effect
본 발명의 생약추출물이 중간선량의 방사선에 의한 조혈계 장해를 방호할 수 있는지를 확인하기 위하여, 비장내 조혈세포의 집락 형성 증가 효과(Endogenous spleen colony formation)를 조사하여 골수의 조혈모세포를 방호할 수 있는지를 관찰하였다. 생물체가 방사선에 피폭되면 혈액 내의 혈구세포가 급격히 소멸되며, 그 후 서서히 골수의 조혈모세포로부터 재생성되어 나온다. 이 때 비장에서 혈구세포가 빠르게 증식하면서 조혈세포 집락을 형성하게 되는데 이러한 원리에 근거하여 하기와 같은 실험을 수행하였다.In order to confirm whether the herbal extract of the present invention can prevent hematopoietic disorders caused by the intermediate dose of radiation, endogenous spleen colony formation in the spleen is investigated to protect the hematopoietic stem cells of bone marrow. Was observed. When the organism is exposed to radiation, blood cells in the blood rapidly die out, and then gradually regenerate from the hematopoietic stem cells of the bone marrow. At this time, hematopoietic cell colonies are formed by rapidly proliferating blood cells in the spleen. Based on this principle, the following experiment was performed.
먼저, 생쥐를 방사선에 피폭시키기 전에 생약추출물을 주사한 다음, 6.5 Gy의 방사선에 노출시켰다. 실험군은 각 군당 8 ∼ 9 마리의 생쥐로 하였으며, 생약추출물의 투여는 생쥐 마리 당 1 ㎎의 용량으로 방사선 조사 전 36 및 12 시간 전에 복강 내로 2회 주사하였다. 방사선 조사후, 9일째에 각 실험군의 생쥐를 희생시키고 비장을 채취하여 보우인(Bouin) 고정액으로 고정한 후 비장 표면에 형성된 조혈세포의 집락을 실체현미경으로 관찰하고 계수하였다.First, the herbal extracts were injected before the mice were exposed to radiation and then exposed to 6.5 Gy of radiation. The experimental group consisted of 8 to 9 mice in each group, and the herbal extract was injected twice intraperitoneally 36 and 12 hours before irradiation at a dose of 1 mg per mouse. On the 9th day after irradiation, the mice of each experimental group were sacrificed, the spleens were collected, fixed with Bowin fixative, and the colonies of hematopoietic cells formed on the spleen surface were observed and counted by a microscope.
그 결과, 비장 표면에 형성된 조혈세포의 집락수가 방사선 피폭 대조군에서는 평균 2.67개, 본 발명의 추출물 투여군에서는 10.13개로 나타나, 상기의 생약추출물이 조혈모세포의 생존수를 현저하게(p<0.05) 증가시킴을 확인하였다(도 8). 이는 본 발명의 생약추출물이 방사선에 의한 조혈계 장해를 방호할 수 있음을 의미한다.As a result, the average number of hematopoietic cells formed on the spleen surface was 2.67 in the radiation exposure control group and 10.13 in the extract-administered group of the present invention. The herbal extract significantly increased the survival number of hematopoietic stem cells (p <0.05). It was confirmed ( Fig. 8 ). This means that the herbal extract of the present invention can protect the hematopoietic system disorder by radiation.
<3-3> 방사선 조사 생쥐에서 혈액세포의 재생성 촉진 효과<3-3> Promoting Regeneration of Blood Cells in Irradiated Mice
본 발명의 생약추출물이 방사선 조사로 인한 조혈기능의 저하를 회복시킬 수 있는지 확인하기 위하여, 본 발명의 생약추출물을 생쥐에 주사한 다음, 방사선을 조사하여 시일경과에 따른 혈액 내의 세포 수를 측정하였다. 생물체가 방사선에 피폭되면 혈액 내의 혈구세포가 급격히 소멸되며, 그 후 서서히 골수의 조혈모세포로부터 재생성되어 나온다. 이 때 혈액 내에 재생성된 혈액세포 수를 측정하여 비교하기 위하여 하기와 같은 실험을 수행하였다.In order to confirm that the herbal extract of the present invention can recover the decrease in hematopoietic function due to irradiation, the herbal extract of the present invention was injected into mice, and then irradiated with radiation to measure the number of cells in blood according to the passage of time. . When the organism is exposed to radiation, blood cells in the blood rapidly die out, and then gradually regenerate from the hematopoietic stem cells of the bone marrow. At this time, the following experiment was performed to measure and compare the number of regenerated blood cells in the blood.
먼저, 본 발명의 생약추출물을 생쥐에 주사한 다음, 4 Gy의 방사선에 노출시켰다. 실험군은 각 군당 5마리의 생쥐로 하였으며, 생약추출물의 투여는 생쥐 마리 당 1 ㎎의 용량으로 방사선 조사 전 36 및 12시간 전에 복강 내로 2회 주사하였다. 방사선 조사 후, 3일, 1주, 2주, 3주, 5주, 7주째에 각 실험군 생쥐의 꼬리정맥을 통해 약 100 ㎕의 혈액을 항응고제가 첨가된 튜브에 채취하여 혈액내 각 혈액세포를 혈구자동분석기(Hemavet 850+)로 계수하였다.First, the herbal extract of the present invention was injected into mice, and then exposed to 4 Gy of radiation. The experimental group consisted of 5 mice in each group, and the herbal extract was injected twice intraperitoneally 36 and 12 hours before irradiation at a dose of 1 mg per mouse. At 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, and 7 weeks after irradiation, approximately 100 μl of blood was collected through the tail vein of each group of mice in a tube containing anticoagulant, and each blood cell in the blood was collected. It was counted by a hematocytometer (Hemavet 850+).
그 결과, 전체 백혈구의 수는 방사선 조사 대조군의 경우 방사선조사 전에 비해 3일째에 약 13% 정도로 급격히 감소하였고, 1 내지 2주 후부터 서서히 회복되기 시작하여 3주 후에 약 62% 정도로 회복하였으며 7주 후에는 정상수준으로 회복되는 경향을 나타내었다. 방사선 조사 전에 본 발명의 생약추출물을 투여한 경우 2주 후 까지는 큰 차이를 보이지 않았으나 3주 후에는 정상수준의 약 85% 정도로 대조군에 비해 유의한 회복효과를 나타내었고, 이러한 효과는 7주까지 지속되었다(도 9). 적혈구 수는 유핵 세포와는 달리 방사선 조사 후 서서히 감소하여 대조군의 경우 2주 후에 정상세포수의 약 56% 까지 감소하였다가 그 후 서서히 회복되어 7주 후에 정상으로 회복되었으나, 방사선 조사 전에 본 발명의 생약추출물을 투여한 경우 극적인 회복효과를 나타내지는 않았지만, 실험기간 중에 방사선 조사군에 비해 높은 세포 수를 나타내어 어느 정도의 회복 효과를 보였다(도 10).As a result, the total number of leukocytes decreased rapidly by about 13% at 3 days compared to before irradiation in the radiation control group, and started to recover slowly after 1 to 2 weeks and then recovered to about 62% after 3 weeks. Showed a tendency to return to normal level. When the herbal extract of the present invention was administered before irradiation, there was no significant difference until 2 weeks, but after 3 weeks, it showed a significant recovery effect compared to the control group at about 85% of the normal level. ( FIG. 9 ). Unlike the nucleated cells, the erythrocyte count gradually decreased after irradiation, and after 2 weeks in the control group, it decreased to about 56% of normal cells, and then gradually recovered and recovered to normal after 7 weeks. When the herbal extracts were administered, they did not show a dramatic recovery effect, but showed some recovery effect due to the high cell number compared to the radiation group during the experiment ( FIG. 10 ).
상기의 결과로부터 본 발명의 생약추출물이 방사선에 의한 조혈계 장해를 방호할 뿐만 아니라 조혈기능의 회복을 촉진시킴을 확인하였다.From the above results, it was confirmed that the herbal extract of the present invention not only prevents hematopoietic disorders caused by radiation, but also promotes recovery of hematopoietic function.
<3-4> 재생조직의 원줄기세포 자사(自死) 억제 효과<3-4> Inhibitory Effect of Regenerated Tissue on Stem Cells
본 발명의 생약추출물이 저선량의 방사선에 의한 장해의 하나인 세포자사(細胞自死, apoptosis)를 억제시킬 수 있는가를 확인하기 위하여 소장 움 세포(intestinal crypt cells)의 자사(自死)를 억제하는지를 관찰하였다. Observe whether the herbal extract of the present invention inhibits intestinal crypt cells in order to confirm whether they can inhibit apoptosis, one of the obstacles caused by low-dose radiation. It was.
실험은 각 군당 4마리씩의 생쥐를 대상으로 하였으며, 생약추출물의 투여는생쥐 마리 당 1㎎의 용량으로 방사선 조사 전 36 및 12시간 전에 복강 내로 2회 주사하였다. 2 Gy의 방사선 조사 후 6시간 지나서 생쥐를 희생시키고 소장을 채취하여 최소 30분 동안 카노이(Carnoy's) 고정액에 고정시킨 뒤, 각 생쥐 마리 당 8 ~ 10개의 소장편을 통상적인 방법에 따라 파라핀 포매하여 횡단절편을 제작하였다. 절편내 소장 움의 세포를 관찰하기 위하여 헤마톡실린-에오신(hematoxylin-eosin)으로 염색하거나 세포자사 과정에서 생성된 DNA 분절(fragments) 측정을 위하여 원발부위 자사(自死) 검출 킷트(in situapoptosis detection kit; APOPTAG TM, Oncor, Gaithersburg, MD, U.S.A.)를 사용한 원발부위 DNA 말단 표지법(in situDNA end-labeling ; ISEL)을 실시하였다. 원발부위 DNA 말단 표지법은 표본 슬라이드에 말단 데옥시 뉴클레오티딜 전달효소(terminal deoxynucleotidyl transferase)를 첨가하여, 분절된 DNA에 디곡시제닌-뉴클레오타이드(digoxigenin-nucleotides)를 부착시키고 항 디곡시제닌 항체-퍼옥시다제(anti-digoxigenin-peroxidase antibody)를 결합시킨 후 디아미노벤지딘(diaminobenzidine ; Sigma Chemical Co.)을 사용하는 통상적인 방법으로 퍼옥시다제(peroxidase) 부위를 발색시켰다. 생쥐 마리당 40개의 소장 움을 광학현미경으로 관찰하였으며, 측정에 사용된 소장 움은 움의 편측 세포 수가 17개 이상으로 파네쓰 세포(Paneth cell)와 내강이 확연히 나타나는 정확히 종절된 움만을 선택하였고, 소장 움의 파네쓰 세포(Paneth cell)를 제외한 4번째 세포까지를 기저부(base)로 하여 자사세포(自死細胞, apoptotic cell)를 기저부와 전체 소장 움에서 관찰되는 총수(total)로 구분하여 계수하였다. 여러 개의 자사체(自死體, apoptotic body)가 그 크기와 형태를고려할 때, 한 세포의 잔유물로 나타날 때는 한 개의 세포로 계수하였다.The experiment was conducted on 4 mice in each group, and the herbal extract was injected twice intraperitoneally 36 and 12 hours before irradiation at a dose of 1 mg per mouse. After 6 hours of irradiation with 2 Gy, the mice were sacrificed and the small intestine was collected and fixed in Carnoy's fixative for at least 30 minutes, followed by paraffin embedding 8 to 10 small intestines per animal. The cross section was produced. In situ apoptosis staining with hematoxylin-eosin to observe the cells of the small intestine in the sections, or in situ apoptosis for measuring DNA fragments generated during cell death. In situ DNA end-labeling (ISEL) was performed using a detection kit (APOPTAG ™, Oncor, Gaithersburg, MD, USA). Primary DNA end-labeling involves the addition of terminal deoxynucleotidyl transferase to sample slides, attaching digoxigenin-nucleotides to fragmented DNA and anti-digoxigenin antibody-per After binding the oxidase (anti-digoxigenin-peroxidase antibody), the peroxidase site was developed by a conventional method using diaminobenzidine (Sigma Chemical Co.). Forty small intestine per mouse were observed under an optical microscope, and the small intestine used for the measurement was selected only for precisely terminated um, which clearly showed Paneth cells and lumen with 17 or more unilateral cells. Apoptotic cells were counted by dividing the total number of apoptotic cells from the basal part and the entire small intestine into the base, with the fourth cell except the Paneth cell of Um as a base. . Considering the size and shape of several apoptotic bodies, they are counted as one cell when appearing as a remnant of one cell.
그 결과,도 11에서 보듯이 소장 움의 기저부에서의 자사 세포 수는 정상 대조군에서는 움 당 평균 0.071개, 방사선 피폭 대조군에서는 2.875개, 생약추출물 투여군에서는 2.056개로 나타났으며, 소장 움 전체에서의 자사 세포 수는 정상 대조군에서는 움 당 평균 0.091개, 방사선 피폭 대조군에서는 3.138개, 생약추출물 투여군에서는 2.456개로 나타났다.As a result, as shown in FIG. 11 , the number of cells in the base of the small intestine was 0.071 per um in the normal control group, 2.875 in the radiation exposure control group and 2.056 in the herbal extract administration group. The number of cells was 0.091 per unit in the normal control group, 3.138 in the radiation exposure control group and 2.456 in the herbal extract group.
상기 결과로부터 본 발명의 생약추출물이 방사선에 의한 세포자사를 억제함을 확인하였으며, 나아가 방사선에 의한 재생조직의 세포 장해를 방호할 수 있음을 확인하였다.From the above results, it was confirmed that the herbal extract of the present invention inhibited cell death caused by radiation, and furthermore, it was confirmed that the cellular damage of regenerated tissues caused by radiation could be protected.
<실시예 4> 생쥐에 대한 경구투여 및 복강투여 급성 독성실험Example 4 Acute Toxicity Test of Oral and Intraperitoneal Administration in Mice
생쥐(C57BL/6 mouse)를 사용하여 본 발명의 생약추출물의 급성독성실험을 실시하였다. 실험군당 6마리씩의 생쥐에 상기 실시예 1의 생약추출물을 최대 10 g/kg의 용량으로 하여 각 용량단계별로 경구투여 및 복강내 주사하였다. 경구투여군은 2주간 관찰하였으며, 복강내주사군은 1주간 관찰하였다. 시험물질 투여후 동물의 폐사여부, 임상증상, 체중변화를 관찰하고 혈액학적 검사와 혈액생화학적검사를 실시하였으며, 부검하여 육안으로 복강장기와 흉강장기의 이상여부를 관찰하고 현미경검사를 실시하였다. 시험결과, 경구투여 군에서는 50% 치사량이 체중 ㎏당 10 g 이상으로 독성이 없었으며, 복강내 주사군에서는 50% 치사량이 체중 ㎏당 수컷의 경우 0.9 g이었으며 암컷의 경우 0.4 g으로 독성효과는 경미하였다. 경구투여군에서는 임상증상, 부검시 육안적 소견 및 현미경 검사 소견에서 정상의 소견을 보였으나, 복강내주사 군에서는 고농도 투여군(수컷 : 1.1 g/㎏ 이상, 암컷 : 0.5 g/㎏ 이상)에서 임상증상으로 비출혈, 안구출혈, 후구마비가 일부 관찰되었고 부검소견에서 장출혈, 신장창백, 신장점상출혈, 간의 백색반, 복수저류가 있었으며 현미경소견상 신장의 사구체충혈, 피질부 세뇨관 공포변성 괴사 및 충출혈, 간장에서 소엽중심성 괴사 및 충출혈, 심근의 변성, 비장의 적색수 충혈, 폐의 충출혈이 관찰되었다.Acute toxicity experiments of the herbal extracts of the present invention were conducted using mice (C57BL / 6 mouse). Six mice per experimental group were orally administered and intraperitoneally injected at each dose level using the herbal extract of Example 1 at a dose of up to 10 g / kg. The oral administration group was observed for 2 weeks, and the intraperitoneal injection group was observed for 1 week. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, 50% lethal dose was more than 10 g / kg body weight in oral administration group, 50% lethal dose was 0.9 g per male body weight / kg in male and 0.4 g in female. It was mild. In the oral administration group, the clinical symptoms, gross and microscopic findings at the time of necropsy were normal, but in the intraperitoneal injection group, the clinical symptoms were higher in the high-dose group (male: more than 1.1 g / kg, female: more than 0.5 g / kg). There were some cases of nasal bleeding, ocular bleeding, and posterior palsy, and autopsy findings included intestinal bleeding, renal paleness, renal bleeding, hepatic white plaque and ascites. In the liver, lobular-centered necrosis and bleeding, myocardial degeneration, splenic red blood clot and lung bleeding were observed.
이상의 결과, 본 발명의 생약추출물은 생쥐에서 경구투여 시 2 g/kg까지 독성변화를 나타내지 않았으며, 복강내주사시 50% 치사량(LD50)이 0.4 g/kg 이상으로 나타나 독성이 경미한 물질로 판단되었다.As a result, the herbal extract of the present invention did not show a toxic change up to 2 g / kg in oral administration in mice, 50% lethal dose (LD 50 ) at 0.4 g / kg or more intraperitoneal injection as a mild toxicity Judging.
상기에서 살펴 본 바와 같이, 본 발명의 생약추출물은 기존의 생물화학적 조혈 및 면역증강제제들보다 부작용(side effect)과 독성이 작고 안전성을 보강한 것으로, 다양한 질병의 예방 및 회복 또는 노약자를 위한 조혈 및 면역기능 증진에 광범위하게 활용될 수 있으며, 방사선 치료시 또는 방사선 피폭 시 조혈계 및 재생조직 원줄기세포의 방호가 일차적인 관건임을 감안하면 방사선 장해의 예방 및 극복에 유용하게 이용될 수 있다. 또한 본 혼합물은 독성이 작은 천연물이므로 본 시료를 바탕으로 한 효능이 높은 조혈기능과 면역기능 증진 기능성 식품의 제조에이용될 수 있다.As described above, the herbal extract of the present invention has less side effects and toxicity and reinforces safety than conventional biochemical hematopoietic and immunopotentiating agents, and prevents and recovers various diseases or hematopoiesis for the elderly. And it can be widely used to enhance immune function, and considering the protection of hematopoietic stem cells and regenerative tissue stem cells during the radiation treatment or radiation exposure is the primary key can be useful in the prevention and overcoming of radiation disorders. In addition, the mixture is a low-toxic natural product can be used in the production of high-efficacy hematopoietic and immune-functional functional foods based on this sample.
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2001-0028743A KR100440863B1 (en) | 2001-05-24 | 2001-05-24 | Extract of herb mixture for heamatopoiesis augmentation and protection from radiation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2001-0028743A KR100440863B1 (en) | 2001-05-24 | 2001-05-24 | Extract of herb mixture for heamatopoiesis augmentation and protection from radiation |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR10-2003-0080989A Division KR100449655B1 (en) | 2003-11-17 | 2003-11-17 | A food for heamatopoiesis augmentation, immunity augmentation and protection from radiation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20020089817A KR20020089817A (en) | 2002-11-30 |
| KR100440863B1 true KR100440863B1 (en) | 2004-07-19 |
Family
ID=27706323
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR10-2001-0028743A KR100440863B1 (en) | 2001-05-24 | 2001-05-24 | Extract of herb mixture for heamatopoiesis augmentation and protection from radiation |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR100440863B1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101350144B1 (en) * | 2013-05-03 | 2014-01-10 | 재단법인 통합의료진흥원 | Compostions for promoting proliferation of bone marrow stem cell comprising palmultang extract |
| WO2015088153A1 (en) * | 2013-12-10 | 2015-06-18 | 한국한의학연구원 | Composition containing sagunjatang as active ingredient for promoting reprogramming of cells into induced pluripotent stem cells, and method for preparing induced pluripotent stem cells using same |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100697212B1 (en) * | 2005-01-29 | 2007-03-22 | 서울향료(주) | Hematopoietic enhancer comprising herbal mixture extract of Astragali Radix and Angelicae Gigantis Radix for treating side-effect caused by administrating anticancer agent |
| US8137710B2 (en) | 2008-12-15 | 2012-03-20 | EcoPharm Corporation | Treating idiopathic thrombocytopenic purpura with comprising extracts of Astragalus membranaceus |
| CN102940702B (en) * | 2012-10-31 | 2014-05-28 | 成都医路康医学技术服务有限公司 | Medicine composition for treating cardia-cerebrovascular diseases |
| KR101652730B1 (en) | 2015-10-08 | 2016-09-01 | 한국원자력연구원 | Method for preparation of herbal composition increased fat-soluble polyphenols, herbal composition prepared thereby, and using thereof |
-
2001
- 2001-05-24 KR KR10-2001-0028743A patent/KR100440863B1/en active IP Right Grant
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101350144B1 (en) * | 2013-05-03 | 2014-01-10 | 재단법인 통합의료진흥원 | Compostions for promoting proliferation of bone marrow stem cell comprising palmultang extract |
| WO2014178675A1 (en) * | 2013-05-03 | 2014-11-06 | 재단법인 통합의료진흥원 | Composition containing palmul-tang extract for promoting proliferation of stem cells derived from bone marrow |
| US10286040B2 (en) | 2013-05-03 | 2019-05-14 | Comprehensive And Integrative Medicine Institute | Composition containing palmultang extract for promoting proliferation of stem cells derived from bone marrow |
| WO2015088153A1 (en) * | 2013-12-10 | 2015-06-18 | 한국한의학연구원 | Composition containing sagunjatang as active ingredient for promoting reprogramming of cells into induced pluripotent stem cells, and method for preparing induced pluripotent stem cells using same |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20020089817A (en) | 2002-11-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20190276803A1 (en) | Method of culturing immune cells, kit for thereof, immune cell cultured medium obtained by same method, cosmetic composition and pharmaceutical composition comprising thereof | |
| KR101834872B1 (en) | Pharmaceutical composition for prevention or treatment of alpha-type herpes virus infection comprising the extract of Elaeocarpus sylvestris or fraction thereof as an active ingredient | |
| JP4055951B2 (en) | Herbal medicine composition having anticancer, immunity and hematopoietic function enhancement effect and oxidative biological damage suppression effect, and production method thereof | |
| KR100440863B1 (en) | Extract of herb mixture for heamatopoiesis augmentation and protection from radiation | |
| KR100310979B1 (en) | Natural pharmaceutical composition for prevention and treatment of hepatic disease. | |
| KR100449655B1 (en) | A food for heamatopoiesis augmentation, immunity augmentation and protection from radiation | |
| KR100401955B1 (en) | The pharmaceutical composition and its preparation method of herb mixture for immunomodulation, heamatopoiesis augmentation and protection from radiation | |
| Subramoniam et al. | Inhibition of antigen-induced degranulation of sensitized mast cells by Trichopus zeylanicus in mice and rats | |
| KR100506396B1 (en) | The pharmaceutical composition and its preparation method of herb mixture for cancer inhibition, recovery of immune system and protection of body from oxidative damage | |
| US11026988B2 (en) | Traditional Chinese medicine composition and use thereof | |
| EP4248964A1 (en) | Pharmaceutical composition for treating sepsis, and use thereof | |
| JP3323765B2 (en) | Cell adhesion inhibitor | |
| KR101999842B1 (en) | Composition for preventing or treating inflammatory disease including extract of adipose-derived stem cells | |
| KR20210097062A (en) | Use of benzene derivatives as immunosuppressive agents | |
| KR101000953B1 (en) | EXTRACTS OF Lespedeza cuneate G. Don AND THEIR USES | |
| KR20150105561A (en) | Pharmaceutical composition for preventing or treating bone disease comprising Hovenia dulcis Thunb extract | |
| KR102160627B1 (en) | Pharmaceutical composition for preventing or treating bone disease comprising extracts of branches of Hovenia dulcis Thunb | |
| US20040076618A1 (en) | Placental preparation having antitumor activity | |
| KR20040087407A (en) | Immune stimulative constituents of ginseng saponins | |
| JPH10158184A (en) | Cell adhesion inhibitor | |
| KR100510429B1 (en) | A mixture extract with immunomodulation and anti-cancer activity and its preparation | |
| TW201726159A (en) | A pharmaceutical composition for treating or alleviating autoimmune-related diseases and use of an active ingredient in the pharmaceutical composition | |
| JP4649617B2 (en) | Pharmaceutical and extract used therefor | |
| Gopalakrishnan et al. | Antihepatotoxic acticity of Coccinia indica | |
| WO2005063255A1 (en) | Pharmaceutical composition for treating and preventing cancer comprising cinnamoni cortex extract and zizyphi fructus extract |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| A107 | Divisional application of patent | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant | ||
| FPAY | Annual fee payment |
Payment date: 20130709 Year of fee payment: 10 |
|
| FPAY | Annual fee payment |
Payment date: 20140630 Year of fee payment: 11 |
|
| FPAY | Annual fee payment |
Payment date: 20150707 Year of fee payment: 12 |
|
| FPAY | Annual fee payment |
Payment date: 20160711 Year of fee payment: 13 |
|
| FPAY | Annual fee payment |
Payment date: 20170620 Year of fee payment: 14 |
|
| FPAY | Annual fee payment |
Payment date: 20190613 Year of fee payment: 16 |