GB2445213A - Flavonoid-rich tissue from Neomarica gracilis - Google Patents
Flavonoid-rich tissue from Neomarica gracilis Download PDFInfo
- Publication number
- GB2445213A GB2445213A GB0715201A GB0715201A GB2445213A GB 2445213 A GB2445213 A GB 2445213A GB 0715201 A GB0715201 A GB 0715201A GB 0715201 A GB0715201 A GB 0715201A GB 2445213 A GB2445213 A GB 2445213A
- Authority
- GB
- United Kingdom
- Prior art keywords
- tissue
- gracilis
- tectorigenin
- flavonoid
- rich
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 240000002374 Neomarica gracilis Species 0.000 title claims abstract description 100
- 229930003935 flavonoid Natural products 0.000 title claims abstract description 78
- 235000017173 flavonoids Nutrition 0.000 title claims abstract description 78
- 150000002215 flavonoids Chemical class 0.000 title claims abstract description 78
- OBBCRPUNCUPUOS-UHFFFAOYSA-N tectorigenin Chemical compound O=C1C2=C(O)C(OC)=C(O)C=C2OC=C1C1=CC=C(O)C=C1 OBBCRPUNCUPUOS-UHFFFAOYSA-N 0.000 claims abstract description 184
- UYLQOGTYNFVQQX-UHFFFAOYSA-N psi-tectorigenin Natural products COC1=C(O)C=C(O)C(C2=O)=C1OC=C2C1=CC=C(O)C=C1 UYLQOGTYNFVQQX-UHFFFAOYSA-N 0.000 claims abstract description 92
- OYUJPVCKGSEYDD-UHFFFAOYSA-N tectorigenin Natural products COc1c(O)cc2OCC(C(=O)c2c1O)c1ccc(O)cc1 OYUJPVCKGSEYDD-UHFFFAOYSA-N 0.000 claims abstract description 92
- 238000000338 in vitro Methods 0.000 claims abstract description 48
- 238000000034 method Methods 0.000 claims abstract description 48
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 230000002062 proliferating effect Effects 0.000 claims abstract description 6
- 239000001963 growth medium Substances 0.000 claims description 53
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 42
- 239000005648 plant growth regulator Substances 0.000 claims description 37
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- 239000002609 medium Substances 0.000 claims description 25
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims description 22
- 239000000725 suspension Substances 0.000 claims description 20
- 239000000203 mixture Substances 0.000 claims description 18
- 150000003839 salts Chemical class 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 15
- HUTDUHSNJYTCAR-UHFFFAOYSA-N ancymidol Chemical compound C1=CC(OC)=CC=C1C(O)(C=1C=NC=NC=1)C1CC1 HUTDUHSNJYTCAR-UHFFFAOYSA-N 0.000 claims description 15
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 claims description 14
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 claims description 14
- 229960001669 kinetin Drugs 0.000 claims description 14
- 239000006870 ms-medium Substances 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 239000000284 extract Substances 0.000 claims description 12
- 229930192334 Auxin Natural products 0.000 claims description 10
- 239000002363 auxin Substances 0.000 claims description 10
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 claims description 10
- 239000004062 cytokinin Substances 0.000 claims description 10
- 239000003617 indole-3-acetic acid Substances 0.000 claims description 10
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 9
- 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 claims description 9
- 229930006000 Sucrose Natural products 0.000 claims description 9
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 claims description 9
- 239000005720 sucrose Substances 0.000 claims description 9
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 8
- 150000001720 carbohydrates Chemical class 0.000 claims description 7
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 7
- 238000007654 immersion Methods 0.000 claims description 7
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical group O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 7
- 229940088594 vitamin Drugs 0.000 claims description 7
- 229930003231 vitamin Natural products 0.000 claims description 7
- 235000013343 vitamin Nutrition 0.000 claims description 7
- 239000011782 vitamin Substances 0.000 claims description 7
- 229960000367 inositol Drugs 0.000 claims description 6
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 6
- 229960003512 nicotinic acid Drugs 0.000 claims description 5
- 229940011671 vitamin b6 Drugs 0.000 claims description 5
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 4
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 4
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 4
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 4
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 4
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 4
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 239000011575 calcium Substances 0.000 claims description 4
- 229910052791 calcium Inorganic materials 0.000 claims description 4
- 229910052802 copper Inorganic materials 0.000 claims description 4
- 239000010949 copper Substances 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 229910052742 iron Inorganic materials 0.000 claims description 4
- 239000011777 magnesium Substances 0.000 claims description 4
- 229910052749 magnesium Inorganic materials 0.000 claims description 4
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 claims description 4
- 235000001968 nicotinic acid Nutrition 0.000 claims description 4
- 239000011664 nicotinic acid Substances 0.000 claims description 4
- 239000011591 potassium Substances 0.000 claims description 4
- 229910052700 potassium Inorganic materials 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 4
- 229910052708 sodium Inorganic materials 0.000 claims description 4
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 claims description 4
- 235000019157 thiamine Nutrition 0.000 claims description 4
- 229960003495 thiamine Drugs 0.000 claims description 4
- 239000011721 thiamine Substances 0.000 claims description 4
- 239000011701 zinc Substances 0.000 claims description 4
- 229910052725 zinc Inorganic materials 0.000 claims description 4
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 3
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 claims description 3
- 229910002651 NO3 Inorganic materials 0.000 claims description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 3
- 150000001447 alkali salts Chemical class 0.000 claims description 3
- 239000010941 cobalt Substances 0.000 claims description 3
- 229910017052 cobalt Inorganic materials 0.000 claims description 3
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 claims description 3
- 229910052750 molybdenum Inorganic materials 0.000 claims description 3
- 239000011733 molybdenum Substances 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 235000008160 pyridoxine Nutrition 0.000 claims description 3
- 239000011677 pyridoxine Substances 0.000 claims description 3
- 229910001868 water Inorganic materials 0.000 claims description 3
- 230000000259 anti-tumor effect Effects 0.000 claims 1
- 238000004445 quantitative analysis Methods 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 description 135
- 230000012010 growth Effects 0.000 description 26
- 230000000694 effects Effects 0.000 description 22
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 21
- 241000196324 Embryophyta Species 0.000 description 20
- 239000002054 inoculum Substances 0.000 description 17
- 239000007788 liquid Substances 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 230000010076 replication Effects 0.000 description 8
- 241000596151 Iris domestica Species 0.000 description 7
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 235000017807 phytochemicals Nutrition 0.000 description 5
- 229930000223 plant secondary metabolite Natural products 0.000 description 5
- 230000008467 tissue growth Effects 0.000 description 5
- 239000004215 Carbon black (E152) Substances 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 229930195733 hydrocarbon Natural products 0.000 description 4
- 150000002430 hydrocarbons Chemical class 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 239000012879 subculture medium Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000002028 Biomass Substances 0.000 description 3
- 101000845005 Macrovipera lebetina Disintegrin lebein-2-alpha Proteins 0.000 description 3
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229910052782 aluminium Inorganic materials 0.000 description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 235000006708 antioxidants Nutrition 0.000 description 3
- 239000011888 foil Substances 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- IYTUKSIOQKTZEG-UHFFFAOYSA-N (3-chloro-4-hydroxyphenyl)acetic acid Chemical compound OC(=O)CC1=CC=C(O)C(Cl)=C1 IYTUKSIOQKTZEG-UHFFFAOYSA-N 0.000 description 2
- HXKWSTRRCHTUEC-UHFFFAOYSA-N 2,4-Dichlorophenoxyaceticacid Chemical compound OC(=O)C(Cl)OC1=CC=C(Cl)C=C1 HXKWSTRRCHTUEC-UHFFFAOYSA-N 0.000 description 2
- GOLXRNDWAUTYKT-UHFFFAOYSA-N 3-(1H-indol-3-yl)propanoic acid Chemical compound C1=CC=C2C(CCC(=O)O)=CNC2=C1 GOLXRNDWAUTYKT-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241001113425 Iridaceae Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 235000011148 calcium chloride Nutrition 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 235000015165 citric acid Nutrition 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000011785 micronutrient Substances 0.000 description 2
- 235000013369 micronutrients Nutrition 0.000 description 2
- 235000010755 mineral Nutrition 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 235000010333 potassium nitrate Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 description 2
- QBYIENPQHBMVBV-HFEGYEGKSA-N (2R)-2-hydroxy-2-phenylacetic acid Chemical compound O[C@@H](C(O)=O)c1ccccc1.O[C@@H](C(O)=O)c1ccccc1 QBYIENPQHBMVBV-HFEGYEGKSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- YKJOCNJSBBEDQC-UHFFFAOYSA-N 3-(3-acetylindol-3-yl)-2-aminopropanoic acid;1h-indole Chemical compound C1=CC=C2NC=CC2=C1.C1=CC=C2C(C(=O)C)(CC(N)C(O)=O)C=NC2=C1 YKJOCNJSBBEDQC-UHFFFAOYSA-N 0.000 description 1
- UOQHWNPVNXSDDO-UHFFFAOYSA-N 3-bromoimidazo[1,2-a]pyridine-6-carbonitrile Chemical compound C1=CC(C#N)=CN2C(Br)=CN=C21 UOQHWNPVNXSDDO-UHFFFAOYSA-N 0.000 description 1
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 229940123457 Free radical scavenger Drugs 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- AZEGJHGXTSUPPG-AWEZNQCLSA-N Indole-3-acetyl-L-valine Chemical compound C1=CC=C2C(CC(=O)N[C@@H](C(C)C)C(O)=O)=CNC2=C1 AZEGJHGXTSUPPG-AWEZNQCLSA-N 0.000 description 1
- 241001479611 Iris ensata Species 0.000 description 1
- 241000822848 Iris nigricans Species 0.000 description 1
- 240000004101 Iris pallida Species 0.000 description 1
- 235000015265 Iris pallida Nutrition 0.000 description 1
- 241001517074 Iris sanguinea Species 0.000 description 1
- 244000140747 Iris setosa Species 0.000 description 1
- 235000000827 Iris setosa Nutrition 0.000 description 1
- 241000344726 Iris spuria Species 0.000 description 1
- 241001136673 Iris spuria subsp. carthaliniae Species 0.000 description 1
- 244000050403 Iris x germanica Species 0.000 description 1
- 235000002971 Iris x germanica Nutrition 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- VAFNMNRKDDAKRM-UHFFFAOYSA-N L-N-(1H-Indol-3-ylacetyl)aspartic acid Chemical compound C1=CC=C2C(CC(=O)NC(CC(=O)O)C(O)=O)=CNC2=C1 VAFNMNRKDDAKRM-UHFFFAOYSA-N 0.000 description 1
- 101710147185 Light-dependent protochlorophyllide reductase Proteins 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- FBDCJLXTUCMFLF-QMMMGPOBSA-N N-(indole-3-acetyl)-L-alanine Chemical compound C1=CC=C2C(CC(=O)N[C@@H](C)C(O)=O)=CNC2=C1 FBDCJLXTUCMFLF-QMMMGPOBSA-N 0.000 description 1
- 229910004619 Na2MoO4 Inorganic materials 0.000 description 1
- 241000597058 Neomarica Species 0.000 description 1
- 244000191705 Neomarica caerulea Species 0.000 description 1
- 241000942915 Neomarica imbricata Species 0.000 description 1
- 240000002591 Neomarica northiana Species 0.000 description 1
- 241000942920 Neomarica rupestris Species 0.000 description 1
- 241000124037 Nephtys paradoxa Species 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 241000208160 Nitzschia capitellata Species 0.000 description 1
- 241000935216 Nothrus silvestris Species 0.000 description 1
- 241000965258 Nymphaea candida Species 0.000 description 1
- 101710193909 Protochlorophyllide reductase, chloroplastic Proteins 0.000 description 1
- 244000046146 Pueraria lobata Species 0.000 description 1
- 235000010575 Pueraria lobata Nutrition 0.000 description 1
- 101710109491 Pyruvate synthase subunit PorA Proteins 0.000 description 1
- 101710109487 Pyruvate synthase subunit PorB Proteins 0.000 description 1
- 101710109489 Pyruvate synthase subunit PorC Proteins 0.000 description 1
- 101710109484 Pyruvate synthase subunit PorD Proteins 0.000 description 1
- IWYDHOAUDWTVEP-UHFFFAOYSA-N R-2-phenyl-2-hydroxyacetic acid Natural products OC(=O)C(O)C1=CC=CC=C1 IWYDHOAUDWTVEP-UHFFFAOYSA-N 0.000 description 1
- 241000589180 Rhizobium Species 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 229930003537 Vitamin B3 Natural products 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003266 anti-allergic effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003217 anti-cancerogenic effect Effects 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- QRZAKQDHEVVFRX-UHFFFAOYSA-N biphenyl-4-ylacetic acid Chemical compound C1=CC(CC(=O)O)=CC=C1C1=CC=CC=C1 QRZAKQDHEVVFRX-UHFFFAOYSA-N 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 150000001868 cobalt Chemical class 0.000 description 1
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000000448 cultured tumor cell Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000032459 dedifferentiation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 108010038795 estrogen receptors Proteins 0.000 description 1
- 102000015694 estrogen receptors Human genes 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 230000002443 hepatoprotective effect Effects 0.000 description 1
- 238000003898 horticulture Methods 0.000 description 1
- 239000003290 indole 3-propionic acid Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229940099563 lactobionic acid Drugs 0.000 description 1
- 235000021374 legumes Nutrition 0.000 description 1
- KNJDBYZZKAZQNG-UHFFFAOYSA-N lucigenin Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.C12=CC=CC=C2[N+](C)=C(C=CC=C2)C2=C1C1=C(C=CC=C2)C2=[N+](C)C2=CC=CC=C12 KNJDBYZZKAZQNG-UHFFFAOYSA-N 0.000 description 1
- 235000021073 macronutrients Nutrition 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 229960002510 mandelic acid Drugs 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- VLAPMBHFAWRUQP-UHFFFAOYSA-L molybdic acid Chemical compound O[Mo](O)(=O)=O VLAPMBHFAWRUQP-UHFFFAOYSA-L 0.000 description 1
- DFPAKSUCGFBDDF-UHFFFAOYSA-N nicotinic acid amide Natural products NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000014571 nuts Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- IWZKICVEHNUQTL-UHFFFAOYSA-M potassium hydrogen phthalate Chemical compound [K+].OC(=O)C1=CC=CC=C1C([O-])=O IWZKICVEHNUQTL-UHFFFAOYSA-M 0.000 description 1
- 235000007715 potassium iodide Nutrition 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 description 1
- 239000011764 pyridoxine hydrochloride Substances 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000016160 smooth muscle contraction Effects 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 238000012090 tissue culture technique Methods 0.000 description 1
- UZKQTCBAMSWPJD-UQCOIBPSSA-N trans-Zeatin Natural products OCC(/C)=C\CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-UQCOIBPSSA-N 0.000 description 1
- UZKQTCBAMSWPJD-FARCUNLSSA-N trans-zeatin Chemical compound OCC(/C)=C/CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-FARCUNLSSA-N 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 235000019160 vitamin B3 Nutrition 0.000 description 1
- 239000011708 vitamin B3 Substances 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 235000020985 whole grains Nutrition 0.000 description 1
- 229940023877 zeatin Drugs 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
- 235000009529 zinc sulphate Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
An in vitro flavonoid-rich rhizome tissue of Neomarica gracilis, which is obtained from a tissue culture preparation of an N. gracilis tissue capable of proliferating, such as a root, a leaf, a basal portion of a leaf, and/or a rhizome. The in vitro flavonoid-rich rhizome tissue of N. gracilis contains tectorigenin, which is distinctively different from the naturally grown rhizome of N. gracilis which contains no tectorigenin. Further provided is a method for cultivating the in vitro flavonoid-rich rhizome tissue, a method for extracting the tectorigenin from the flavonoid-rich rhizome tissue, and quantitative methods for determining the amount of tectorigenin in the in vitro flavonoid-rich rhizome tissue.
Description
FLAVONOrDS-RICH TISSUES FROM NEOMARICA GRACILIS AND METHODS
FOR CULTURING THE SAME
RELATED APPLICATION
This patent application claims the priorities of Taiwan Patent Application No. 95149939, filed on December 29, 2006, and U.S. Patent Application No. 11/797, 145, filed on May 1, 2007, which are herein incorporated by reference.
FIELD OF THE INVENTION
This invention relates to an in vitro flavonoid-rich rhizome tissue of Neomarica gracilis, which is provided by a tissue culture preparation using an N. gracilis tissue capable of proliferating, such as a root, a leaf, a basal portion of a leaf, and/or a rhizome.
The flavonoid-rich rhizome tissue of N. gracilis contains tectorigenin, which is distinctively different from the wild rhizome of N. gracilis, which contains no tectorigenin.
The present invention further provides a method for cultivating the flavonoid-rich rhizome tissue in vitro, a method for extracting tectorigenin from the flavonoid-rich rhizome tissue, and quantitative methods for determining the amount of tectorigenin in the flavonoid-rich rhizome tissue.
BACKGROUND OF THE INVENTION
Phytochemicals are substances that plants naturally produce to protect themselves against bacteria, viruses, and fungi. There has been a lot of interest in phytochemicals recently because many of them have demonstrated effects on slowing the aging process and reduce the risk for cancer, heart disease, and other chronic health conditions.
More than 900 different phytochemicals have been found in plant foods, with others still to be discovered. Fruits, vegetables, whole grains, soy and nuts are all sources of these disease-fighting substances. Phytochemicals are usually related to plant pigments,
I
so fruits and vegetables with bright colors (yellow, orange, red, blue, purple, green) contain the most.
Flavonoids are a group of phytochemicals that have long been recognized to possess anti-inflammatory, antioxidant, antiallergic, hepatoprotective, antithrombotic, antiviral, and anticarcinogenic activities. The flavonoids are typically phenolic compounds and, therefore, act as potent metal chelators and free radical scavengers. They are powerful chain-breaking antioxidants. The flavonoids display a remarkable array of biochemical and pharmacological actions, some of which suggest that certain members of this group of compounds may significantly affect the function of various mammalian cellular systems.
Recent reports indicate that plant flavonoids cause the activation ofbacterial (Rhizobium) modulation genes involved in control of nitrogen fixation, which suggests important relationships between particular flaionoids and the activation and expression of mammalian genes (See e.g., Midddledton et al., Pharmacological Reviews, 2000, 52:673-751).
Tectorigenin is a flavonoid. In recently years, development has been made in exploring the potential utility of tectorigenin, which has demonstrated anti-bacterial, anti-inflammatory, and cancer-preventing activities. Moreover, it has been shown that tectorigenim stimulates the production of prostaglandin, induces proliferation of macrophages, selectively modulates the activity of estrogen receptors, and controls smooth muscle contraction.
Tectorigenin is typically extracted from rhizomes of Indaceae plants, such as Iris germanica L., Iris pallida Lam, Iris nigricans, Iris ensata, Iris sanguinea, Iris setosa, and Belamacanda chinensis (B. chinensis). The tectorigenin cOntent in the rhizomes are affected by the growth conditions, such as temperature and humidity. In the case of B. chinensis, it usually takes 2-3 years of growth before the rhizomes can be harvested for tectorigenin extraction. Therefore, there still exists a need for methods that can effectively grow plants with a high tectorigenin content.
Neomarica gracilis (N. gracilis) is a very common horticulture plant that belongs to the Iridaceae family. It can be cultured in large scale at low cost. However, the rhizomes of naturally grown N gracilis does not contain tectorigenin.
In the invention to be presented in the later sections, an in vitro rhizome of N. gracilis is obtained from a tissue culture preparation, which can be harvested in about 1 or 2 months. The in vitro rhizome of N. gracilis is rich in flavonoids and contains high content of tectorigenin, which can be used as a source for tectorigenin.
SUMMARY OF THE INVENTION
The present invention provides an in vitro flavonoid-rich tissue of Neomarica gracilis, which is prepared in a tissue culture environment which alters the flavonoid content of the wild-type N gracilis. In particular, the flavonoid-rich tissue of the present invention contains a substantial amount of tectorigenin.
The in vitro flavonoid-rich tissue of N. gracilis is preferably a rhizome tissue, which is cultured from an N. gracilis tissue capable of proliferating. Examples of the N. gracilis tissue capable of proliferating include the root, the rhizome, the leaf, and a basal portion of the leaf of N. gracilis.
A culture medium is used in the tissue culture for preparing the flavonoid-rich tissue of N. gracilis. This culture medium contains (1) a plant growth regulator, (2) a salt medium, and (3) a carbohydrate. The plant growth regulator (PGR) includes cytokinins or auxins. Examples of the PGRs include, but are not limited to, indole-3-acetic acid, 2-4-dichlorophenoxyacetic acid, a-naphthaleneacetic acid, 6-benzyl-aminopurine, kinetin, and/or a mixture thereof. The plant growth regulator is preferred to be at a concentration of about 0.01 to 2.0 mg/L. The preferred salt medium is a Murashige and Skoog basic salt medium (i.e., an MS medium), which includes, but is not limited to, the following salts: sodium, potassium, nitrate, ammonium, magnesium, sulfate, calcium, iron, chloride, phosphate, manganese, iodine, borate, zinc, copper, molybdenum, cobalt, or a mixture thereof. Examples of the carbohydrate include myo-inositol, sucrose or a mixture thereof.
Optionally, the culture medium can contain a vitamin, such as thiamine HC1, pyridoxine HCI, and nicotinic acid, and an ancymidol. The culture medium has a pH of about 5 to 7.
The tissue culture preparation includes a flask culture, preferably under shaking condition, a Temporary Immersion System (TIS), or a combination thereof.
The flavonoid-rich tissue from N. gracilis contains tectorigenin in the amount of about 2.5 to 65 mg per Kg of dry tissue weight. The total amount of flavonoids after 8 weeks of a solid tissue culture in an MSO (i.e., an MS medium with no plant growth regulator added) is in the amount of about 10.74 mg (based on measurement using -tectorigenin as a standard) per g of dry weight of the flavonoid-rich tissue.
The present invention further provides a method for obtaining the in vitro flavonoid-rich tissue from N. gracilis. The method includes (1) inoculating an N. gracilis tissue in a culture medium of the tissue culture; and (2) growing the N. gracilis tissue in the tissue culture for a sufficient amount of time to allow a rhizome tissue to form. The N. gracilis tissue is capable of cell replication, such as a root, a leaf, a basal portion of a leaf, or a rhizome.
The culture medium is preferably maintained at about 20 C to 30 C.
The tissue culture is a flask culture, a temporary immersion system (uS), or a combination thereof.
The sufficient amount of time for forming the flavonoid-nch rhizome tissue of N. gracE/is is about 4 to 8 weeks.
The TIS allows the N. gracilis tissue to be immersed in the culture medium for about 1-3 minutes in about every 2-4 hours.
The present invention further provides a method for extracting the tectorigenin from the flavonoid-rich tissue of N. gracilis. The method comprises: (1) drying the in vitro flavonoid-rich tissue from N. grad/is to obtain a dried flavonoid-rich tissue; (2) adding an alcohol to the dried flavonoid-rich tissue to form a suspension; (3) heating the suspension to form a heated suspension; and (4) filtering the heated suspension after the heated suspension has cooled off to collect an filtrate which contains tectorigenin. The preferred method to dry the in vitro flavonoid-rich tissue is by freeze-drying. The preferred way to heat the suspension is using ultrasonic vibration while heating the suspension at about 50-70 C for about one hour. Examples of the alcohol which can be added to the dried in vitro flavonoid-rich tissue is methanol or ethanol. Optionally, the dried in vitro flavonoid-rich tissue can be ground before the alcohol is added. The filtrate is preferred to be collected by passing the heated suspension through a Whatman No. 1 filter.
The total amount of tectorigenin extracted from the flavoid-rich tissue of N. gracilis is determined by a high performance liquid chromatography (HPLC). The preferred column for HPLC isa CosmosiRE 5 C 18-AR-I! column. The preferred eluting solution is a solution containing methanol and water (with 0.1% acetic acid) in a volume ratio of 55: 45.
The amount of tectorigenin is measured at about 265 nm wavelength using an tp-tectorigenin (Sigma T-9 165) as a standard.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 is a drawing showing the Temporary Immersion System, which is one of the two tissue culture systems (the other tissue culture system is a flask culture system) used in the present invention.
Figure 2 is a diagram showing the effect of inoculum weight ratio on the growth rate of the in vitro flavonoid-rich rhizome tissue of N. gracilus in a flask culture. Data were collected from the rhizome tissue cultured in an MS medium (Murashige and Skoog medium) containing 0.5 mg/L of 6-benzyl-aminopurine (BA), 0.1 mgTL of 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.84 mgfL of a-naphthaleneacetic acid (NAA) for 3 weeks. Each data point represents an average of 3 replications and the error bars represent the standard errors of the means.
Figure 3 is a diagram showing the effect of indole-3-acetic acid (IAA) and Kinetin on increase of the biomass in the in vitro flavonoid-rich rhizome tissue of Neomarica gracilis, where the rhizome tissue is obtained from a flask culture. Each data point represents an average of 3 replications and error bars represent the standard errors of the means. The biomass increase = current fresh weight -the fresh weight of 2 weeks ago.
Figure 4 is a diagram showing the growth curve of N. gracilis cultured in an MS medium containing 0.1 mg/L (*) of c*naphthaleneacetic acid (NAA) for 8 weeks. Each data point represents an average of 3 replications and error bars represent the standard errors of the means.
Figure 5 is a diagram showing the tectorigenin content of the flavonoid-rich rhizome cultured in 0.1 mgfL (0) of MS + NAA, or lmgfL of NAA and 0.1 mg/L of 2,4-D (U) for 8 weeks. Each data point represents an average of 3 replications and error bars represent the standard errors of the means.
Figure 6 is a composite of pictures showing sprout formation of N. gracilis rhizome tissue in the presence of ancymidol. Photographs were taken after three weeks of culture. Bar = 1 cm. Panel (a): 0.5 mg(L ancymidol added on day 7; Panel (b): 2.0 mg/L ancymidol added on day 7; Panel (C): 2.0 mg/L ancymidol added on day 14, and Panel (d) control culture without any ancymidol.
Figure 7 is a diagram showing the effect of inoculum weight and the plant growth regulators (PGRs) on the growth rate of N. gradiis rhizome tissue. Each data point represents the average of 3 replications and error bars represent the standard errors of the means. The growth rate (Final F.W.-Initial F.W.)/Initial F.W.
DETAILED DESCRIPTION OF THE INVENTION
One aspect of the present invention relates to a flavonoid-rich tissue of N. gracilis, which is obtained from a tissue culture of an N. gracilis tissue capable of proliferation and cell replication, such as root, leaf, the basal portion of a leaf, and rhizome. The flavonoid-rich tissue is an in vitro rhizome tissue, which is distinctively different from a wild rhizome of N gracilis, by containing tectorigenin. Recent research has demonstrated that tectorigenin possesses medicinal effects on eliminating free radicals, halting the progression of tumors, and inhibiting pathological bacteria and fingus, such as H. pylon and mold, etc. Tectorigenin has the chemical structure as shown below:
MOXXQ
Tectorgenin Tectorigenin is wildly found in rhizomes or roots of many Iris and legume plants, such as Iris spuria, Iris carthaliniae, and Iris gerinanica, and Pueraria thunbergiana. However, the amounts of tectorigenin in these plants are too low to be used of sources of tectorigenin.
The main source of tectongenin comes from the naturally grown from the rhizomes of Belamcanda chinensis. However, B. chinensis has a slow growth rate (i.e., from seeding to harvest) of about two to three years, which makes the collection of tectorigenin difficult to achieve.
Contrary to B. chinensis, N. gracilis is a popular semi-outdoor plant, which is easy to grow and propagate. However, N. gracilis does not contain tectongenin. N. gracilis belongs to the family of Iridaceae and the genus of Neomarica, which contains 16 species: N. caerulea, N. capitellata, N. caulosa, N. fiuminensis, N. gracilis, N. imbricata, N longfo1ia, N. nitida, N. northiana, N. paradoxa, N. portosecurensis, N. rotundata, N. rupestris, N. sabini, N. silvestris, and N. variegafa. N. gracilis is native to tropical regions of western Africa, and Central and South America, with the highest concentration in Brazil. N. grad/is is a herbaceous perennial plant that propagate by way of a thick rhizome and new plantlets that develop from the stem where flowers once emerged.
Another aspect of the present invention relates to a method for growing an In vitro rhizome tissue of N. gracilis with high tectorigenin contents by tissue culture techniques for a relatively short period of time, generally between about 4 to 8 weeks and hence provides a new source of tectorigenin. The method comprises the steps of inoculating an N. gracilis tissue in a culture medium, and growing the N. gracilis tissue for a sufficient Oamount of time until the rhizome tissue is developed.
The N gracilis tissue may be any tissue obtained from a naturally grown N. gracilis or a cultured N. gracilis that is capable of proliferation. Preferably, the N. gracilis tissue is a tissue taken from the root, rhizome, leaf, or the basal portion of the leaf of N. gracilis.
The culture medium comprises at least one plant growth regulator (PGR), one salt, and one carbonhydrate. PGRs cause or foster differentiation or dedifferentiation of the explained tissues being propagated in the culture chamber. Examples of PGRs include, but are not limited to, auxins and cytokinins.
Auxin is the active ingredient in most rooting mixtures. Auxin helps the vegetative propagation of plants. On a cellular level, auxins influence cell elongation, cell division and the formation of adventitious roots. Some auxins are active at extremely low concentrations. Auxins may be used in a concentration range of 0.0001-20 mgfL, preferably 0.01-10 mg/L, more preferably 0.01-2.0 mgIL. Examples of auxins include, but are not limited to, 4-Biphenylacetic acid, 3-Chloro-4-hydroxyphenylacetic acid, 4- I-Iydroxyphenylacetic acid, Indole-3-acetic acid (IAA), Indole-3-propionic acid, Indole-3- butyric acid, Indole-3-acetyl-L-alanine, Indole-3-acetyl-DL-aspartic acid, Indole-3-acetyl-DL-tryptophan, Indole-3-acetyl-L-valine, 2,4-dichlorophenoxyacetic acid (2,4-D), and alpha-naphthaleneacetic acid (NAA).
Cytokinins promote cell division, stimulate shoot proliferation, activate gene expression and metabolic activity in general. At the same time, cytokinins inhibit root formation. This makes cytokinins useful in culturing plant cell tissue where strong growth without root formation is desirable. In addition, cytokinins slow the aging process in plants. Cytokinin may be used in a concentration range of 0.0001-20 mgIL, preferably 0.01-10 mg/L, more preferably 0.01-2.0 mgfL. Examples of cytokinins include, but are not limited to, N-(3-methylbut-2-enyl)-1H-punn-6-amine, 6-benzyl-aminopurine (BA), kinetin, and zeatin.
Examples of the salt include salts of inorganic acids, such as nitric acid, hydrochloric acid, phosphoric acid, sulfuric acid, boric acid, iodion acid; organic acids such as acetic acid, malonic acid, mandelic acid, oxalic acid, lactic acid, lactobionic acid, fumaric acid, maleic acid, tartaric acid, citric acid, ascorbic acid. The salt can bc sodium, potassium, calcium, ammonium, iron, magnesium, manganese, zinc, copper, or cobalt salts. The culture medium may contain a mixture of salts.
In one embodiment, the culture medium includes nutrients that foster growth of an explanted plant tissue, such as, for example, the macro-and micronutrients set forth in Murashige & Skoog, Physiol. Plant., 15, 473-497 (1962), which are hereinafter referred to as the "MS basal medium." The salts contained in the MS basal medium is referred to as "MS salts." MS salts used in the context of the present invention include suitable concentrations of ammonium nitrate, boric acid, calcium chloride, cobalt chloride, cupric sulfate, Na2-EDTA, ferrous sulfate, magnesium sulfate, manganese sulfate, molybdic acid, potassium iodide, potassium nitrate, potassium phosphate monobasic, sodium nitrate, sodium phosphate monobasic and zinc sulfate.
The hydrocarbon can be any hydrocarbon that has nutritional value to a plant culture. Preferably, the hydrocarbon is a saccharide. More preferably, the hydrocarbon is myo-inositol, sucrose, or a mixture thereof.
The culture medium may additionally contain other components such as amino acids, vitamins, or mixtures thereof. Preferred vitamins include, but are not limited to, vitamin B! (thiamine HC1), vitamin B6 (pyridoxine HCL), and vitamin B3 (nicotinic acid).
The culture medium is adjusted to a pH range suitable for the growth of N. gracilis.
The pH range is preferably between 4 and 8, and more preferably between 5 and 7. In one embodiment, the culture medium includes suitable buffering agents for maintained the pH at the desired level. These agents will typically have a pKa between about 4.5 and about 5.5, and include, but are not limited to, citric acid, N-morpholino-ethansulfonic acid, potassium hydrogen phthalate, and benzoic acid.
The temperature of the culture is usually maintained at or below about 30 C., preferably within the range of about 20-30 C.
The desired growth period is 2 to 16 weeks, preferably 4-8 weeks.
In a preferred embodiment, the culture medium comprises MS salt, sucrose (30 gIL), myo-inositol (100 mgfL), 6-benzyl-aminopurine (BA) (0.5 mgfL), 2,4-dichrophenoxyacetic acid (2,4-D) (0.1 mg/L), and ct-naphthaleneacetic acid (NAA) (0.g4 mg/L).
In another embodiment, the N. gracilis tissue is cultured with constant agitation. In another embodiment, the N. gracilis tissue is cultured in a Temporary Immersion System ii (TIS). As shown in Figure 1, a TIS nourishes and oxygenates plant cultures by intermittent immersion the plant tissue in the culture medium. Briefly, a chamber 10 holds cultures 12 on a screen 14 or in a basket. Low pressure air is pumped into the chamber 10, forcing the liquid medium 16 up and bathing the cultures 12. The air flow also oxygenates and agitates the medium 16. When the flow is turned off, pressure stops, and the medium 16 returns to the bottom of the chamber 10. Typically, all components of the TIS are autoclavable and reusable. The system may be easily automated for large scale plant tissue culture. In a preferred embodiment, the N. gracilis tissue is immersed by the culture medium for 1-3 minutes every 2-4 hours.
Another aspect of the present invention provides cultured N. gracilis tissues with high levels of tectorigenin. In one embodiment, the cultured N gracilis rhizome tissues have a tectorigenin content of 2.5 - 65 mg I kg dry rhizome weight. The cultured N. gracilis rhizome tissues have a total flavonoids content of 10.741 0.311 (means S.E.) mglg of dry rhizome weight in solid culture containing an MS medium with no FOR added (MSO), which is measured using t'-tectorigenin as a standard.
Another aspect of the present invention relates to a method for extracting flavonoids from N. gracilis tissues. The method includes the steps of drying the tissue, grinding the dried tissue, suspending the ground tissue in an alcohol, heating the suspension, and filtering the suspension to obtain extracts of flavonoids.
The plant tissue may be dried with any method, preferably by freeze-drying using a refrigerated vacuum drier. The alcohol may be any alcohol, preferably methanol, or ethanol. The alcohol is preferably heated to a temperature range of 50 C to 70 C, and more preferably heated with shaking, vibration, or sonication. A preferred method of vibration is ultrasonic wave vibration.
The following experimental designs and result are illustrative, but not limiting the scope of the present invention. Reasonable variations, such as those occur to reasonable artisan, can be made herein without departing from the scope of the present invention.
Also, in describing the invention, specific terminology is employed for the sake of clarity.
However, the invention is not intended to be limited to the specific terminology so selected. It is to be understood that each specific element includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.
EXAMPLES
Example 1: Material and Methods In vifro Flavonoid-rich Rhizome Tissue from N. gracilis (Neomarica gracilis) The in vitro flavonoid-rich rhizome tissues of N. gracilis were cultured from Tatung University Plant Tissue Culture Lab. The rhizome tissues were subcultured every month, with an MS basal salt medium supplemented with 30 g/L sucrose, 100 mgIL myo-inositol, 0.5 mgfL BA, 0.1 mgfL 2,4-D and 0.84 mgfL NAA.
Culture Medium The liquid culture medium comprised the MS basal salt medium (Murashige and Skoog, 1962) (Table 1) supplemented with 30 gIL sucrose, 100 mg/L myo-inositol, and various plant growth factors according to different experiments. The solid culture medium was prepared by adding 8 g/L agar to the liquid medium. The solid culture was carried out in 8 x 1.5 x 1.5 cm culture tubes (10 ml culture medium I tube) or flasks (30 ml culture medium / flask). Liquid culture was carried out in 50 ml, 100 ml or 250 ml flasks with 10 ml, 50 ml or 100 ml culture medium, respectively. The opening of the culture tubes or flasks was covered with aluminum foil.
The TIS culture used the Plantima system from the A-Tech Bioscientific Co. Ltd. About 200 ml medium was added to each culture chamber. Plastic tubings and sterile filters membranes were placed at the gas inlet and outlet as instructed. The filter membrane was wrapped with cotton and aluminum foil, and the plastic tubings were immobilized with clamps to prevent water vapor from entering the filter. The culture chamber was covered with aluminum foil.
All the culture medium had a pH value of 5.70 0.05, and were sterilized by autoclaving at 121 C, under 1.1-1.2 kg/ cm2 pressure, for 15 minutes.
Table 1. The basal salts composition of MS (Murashige and Skoog, 1962) Chemical mg/I Macronutrients KNO3 1900 NH4NO3 1650 MgSO4. 7H20 370 CaCl2. 2H20 440 KH2PO4 170 Micronutrients MnSO4 4H20 22.3 KI 0.83 H3B03 0.2 ZnSO4. 7H20 8.6 CuSO4. 5H20 0.025 Na2MoO4. 2H20 0.25 CoCI2. 6H20 0. 025 FeSO4. 7HO 27.8 Na2EDTA 37.3 Flask culture (1) To determine the effect of inoculum weight (expressed as a percentage of grams of fresh weight of the inoculum / 100 ml of culture medium), liquid cultured N. gracilis rhizome tissue was inoculated to a subculture medium containing an MS basal salt, 0.5 mg/L BA, 0.1 mgIL 2, 4-D and 0.84 mg/L NAA in an inoculurn weight ratio of 0.5, 1, 1.5, 2, 3, 4, 5, 10 or 15 % (g fresh weight (F.W.) / 100 ml medium). The cultured tissue was weighted three weeks after inoculation.
(2) To determine the effect of plant growth regulators (PGR), sprouts from liquid cultured N. gracilis rhizome tissue were removed with a scalpel. The rhizome tissue was inoculated at an inoculum weight of 3% (g F.W. / 50 ml) in culture medium containing MS basic medium supplemented with PORs as shown in Table 2. The culture medium was changed every two weeks. The cultured tissue was weighted at each medium change for a total of eight weeks.
(3) To determining the growth curve of N gracilis rhizome tissue and the tectorigenin content, sprouts from liquid cultured N. gracilis rhizome tissues were removed with a scalpel. The rhizome tissue was inoculated at an inoculuni weight (g F.W. / 50 ml) in culture medium containing the MS basic medium supplemented with 0.1 mg/I. NAA or a mixture of 1 mg1L NAA and 1.0 mg/L 2,4-D. The tissues were weighted and analyzed for tectorigenin content every week for eight consecutive weeks.
(4) To determine ancymidol's effect on sprout formation and tectorigenin content, liquid cultured N. gracilis rhizome tissue was inoculated at an inoculum weight of 3% (g F.W./ 100 ml) to a subculture medium containing MS, 0.5 mgfL BA, 0.1 mg/L 2, 4-D and 0.84 mg/L NAA. At day 7 and 14 of the culture, sterilely filtered (0.2 urn) ancymidol was added to the culture medium to a final concentration of 0.5, 1.0, or 2.0 mgfL. The growth of the tissue was monitored and the tectorigenin content analyzed.
Table 2. Effect of plant growth regulators on N. gracilis tissue growth in flask culture Test Mineral Oqani Substan Plant Growth Regulator (mgfL) Sucrose -sample Comp. v NA lvlI NAA 2,4-D Kinetin IAA (g/L) MSO MS 1 0.5 0.5 2 100 0 0 0 0 30 5.7 Al MS 1 0.5 05 2 100 _____ _____ 0.1 0 30 5.7 A2 MS I 03 0.5 2 100 ____ ____ 1.0 0 30 5.7 Bl MS 1 0.5 0.5 2 100 _____ _____ 0 0.1 30 5.7 02 MS 1 0.5 0.5 2 100 _____ _____ 0.1 0.1 30 5.7 83 MS 1 0.5 0.5 2 100 _____ _____ 1.0 0.1 30 5.7 Cl MS 1 05 05 2 100 _____ _____ 0 0.5 30 5.7 C2 MS = öT T W0 __ __ 0.1 0.5 30 TT C3 MS 0.5 0.5 2 100 _____ _____ 1.0 0.5 30 5.7 Dl MS -05 05 2 100 ____ ____ 0 1.0 30 53 D2 MS -0.5 0.5 2 100 _____ ____ 0.1 1.0 30 5.7 D3 MS -03 05 2 100 ____ ____ 1.0 1.0 30 El MS 0.5 0.5 2 100 _____ _____ 0 2.0 30 5.7 E2 MS -0.5 05 2 100 _____ _____ 0.1 2.0 30 5.7 E3 MS 0.5 0.5 2 100 _____ _____ 1.0 2.0 30 5.7 Fl MS -öT öT 1 100 0 0.1 ______ _____ 30 F2 MS -0.5 05.2 100 0 1.0 ______ _____ 30 5.7 Gi MS 0.5 0.5 2 100 0.1 0 _______ _____ 30 5.7 02 MS -05 0.5 2 100 0.1 0.1 _____ ____ 30 53 03 MS 03 03 2 0 0.1 1.0 ____ ___ 30 53 HI MS 0.5 0.5 2 100 0.5 0 _______ _____ 30 5.7 H2 MS -03 0.5 2 100 0.5 0.1 _____ ____ 30 53 113 MS -0.5 0.5 2 100 0.5 1.0 _______ _____ 30 5.7 II MS --05 0.5 2 100 1.0 0 ______ _____ 30 5.7 12 MS I -0.5 0.5 2 100 1.0 0.1 _______ _____ 30 5.7 [3 MS I 0.5 0.5 2 100 1.0 1.0 ______ _____ 30 5.7 31 MS 1 05 0.5 2 100 2.0 0 _______ _____ 30 5.7 12 MS 0.5 05 2 100 2.0 0.1 _______ _____ 30 5.7 33 MS 1 0.5 0.5 2 100 2.0 1.0 _______ _____ 30 5.7 TIS culture TIS Plantima culture system was purchased from A-Tect Bioscientific Co., Ltd., in Taipei, Taiwan).
(1) To determine the effect of the inoculation weight on tissue growth, the uS culture chamber was' divided into four areas. Liquid cultured N grad/is rhizome tissue was inoculated at 1.5, 3, 6 and 9g in each area. The culture medium was an MS medium supplemented with PGR as described in samples T12, T17 and TiS of Table 3. The tissues were cultured under conditions detailed in the Plantima user's manual and immersed for two minutes every three hours. The cultured tissue was weighted and the medium replaced every ten days.
(2) To determine the effect of PLRs on the grows of N. gracilis and the tectorigenin content influence, ten pieces of liquid cultured N. gracilis rhizome tissue were inoculated in culture media containing MS and PGRs described in samples MSO, T13, T14, T15 and T16 of Table 3. The culture media were replaced on week 2 and the tissues were harvested on week 4 and analyzed for tectongenin content.
Table 3. Effect of plant growth regulators on N. gracilis tissue growthin TIS culture Test Mineral anicSubstanc L Plant Growth Re4atormg Sucrose -sample Comp. v j NA Gly Mi BA NAA 2,4-0 Kinetin (g/L) pH MSO MS 0.5 0.5 2 100 0 0 0 0 30 5.7 112 MS -0.5 05 2 100 0 0 0 1.0 30 S.7 113 MS -0.5 0.5 2 100 0 0.1 0 0 30 5.7 114 MS -0.5 0.5 2 100 0 0.5 0 0 30 T15 MS -05 0.5 2 100 0.5 0.84 0.1 0 30.5*L 116 MS -0.5 0.5 2 100 0 1.0 0.1 0 30 5.7 117 MS -05 0.5 2 100 0' 1.0 1.0 0 30 5.7 118 MS 0.5 0.5 2 100 0 2.0 1.0 0 30 5.7 Extraction of tectorz-enin and total flavonoid N. graci/is rhizome tissue was frozen at -SOC for 10 hours, dried in a refrigerated vacuum drier (VirTis Freezemobile I2XL) overnight, and grinded. 0.5 g of dried and grinded tissue was suspended in adequate amount of methanol in a 10 ml flask, incubatedgrinded tissue was suspended in adequate amount of methanol in a 10 ml flask, incubated at 60'C with ultrasonic vibration for 1 hour, and cooled. Methanol was then added to the suspension to a final volume of 10 ml. The suspension was filtered with Whatman No. I filter paper. The filtered solution was sealed in brown sample vials and placed in cold room for future experiments.
Prior to the determination of tectorigenin content, tectorigenin standard (Sigama T- 9165, V'-tectorigenin) solution and the samples were filtered through a 0.45 urn filter. The filtered samples were sealed in 2 ml brown HPLC vials for further analysis.
Measurement of totalfiavonoid Total flavonoid was measured according to Lee et al., J. Agric. Food Chem. (2003) 51: 7292-7295. Briefly, 4 ml of deionized water and 0.3 ml of 5% NaNO2 were added to about 0.5 ml of N. Gracilis extract to form a sample mixture and allowed for reaction for about 5 minutes. About 0.3 ml of 10% AlCl3 was then added to the reacted sample mixture and allowed for further reaction for an additional 5 minutes under thorough shaking. This was followed by the addition of 2 ml of I N NaOH and 2.9 ml of deionized water to the further reacted sample mixture. The total flavonoid in the resulting reacted sample mixture was then determined by measuring the absorbance at 510 inn wavelength with an UV spectrophotometer (Ultrospec 2000, Pharmacia Biotech), and comparing the data with the standard curve using -tectotigenin as a standard.
Measurement of tector&Q-enin by HPLC The tectorigenin content of the tissue extract was determined by HPLC analysis using a Cosmosil 5 C18-AR-II column (5p m, 4.6x 250 mm), a Lichrospher 100 RP- 18e Guard column (45x 4.6mm, 5 m, Merck), a Degasser ( ERC-3415a) Pump, a Waters 600E Autosampler and Injector (Schambeck SGD GmbH S5200), a Waters TM 486 UV/VIS Detector, and an Integrator (SISC Xunhua Ltd.). The analysis was performed using a methanol: H20 (0.1 % Acetic acid) ratio of 55: 45, a flowrate of 0.8 mIt mm, and a detection wavelength of 265 am. The tectorigenin peak of the standard appeared at about 13 mm appear.
Statistic analysis All the measurements were repeated for three times. The experimental data were analyzed using Duncan's multiple range test with 5 % significance level (Duncan D. B. 1955, Biometrics, 11:1-42).
Morpholov observation and photogrqph Morphology observations were made under a dissecting microscope (Olympus SZ-ET). Photographic records were made using a digit camera (Nikon coolpix 8700).
Example 2: Effect of inoculum weight on tissue growth in flask culture Liquid cultured N. gracilis rhizome tissue was inoculated to a subculture medium containing MS, 0.5 mg/L BA, 0.1 rngul 2, 4-D and 0.84 mg/L NAA at an inoculum weight of 0.5, 1, 1.5,2, 3, 4, 5, 10 or 15 % (g fresh weight (F.W.) /100 ml medium). As shown in Figure 2, the inoculum weight of 3% (g fresh weight (F.W.) / 100 ml medium) resulted the highest growth rate, i.e., about a four fold increase of the fresh weight ((fold increase of the fresh weight = final fresh weight -initial fresh weight) / initial fresh weight). The weight gain diminished when inoculum weight is greater than 4%, probably due to the limited space and nutrition supply in the flask.
Example 3: Effect of PGR on tissue growth and tectorigenin content in flask culture N. gracilis rhizome tissue was inoculated at an inoculum weight of 3% (g F.W./ lOt) ml) in culture medium containing MS supplemented with various amount of PGRs (Table 2). The culture medium was changed and the fresh weight of the tissues was measured every two weeks. Significant increases in fresh weight were observed during 6-8 weeks of culture (Figure 3).
Among the 15 Kinetiri/IAA combinations listed in Table 2, only 4 combinations (i.e., B 1, Cl, C2, and Dl) resulted in significant increases in tectorigenin content. Among the 4 combinations that led to increased tectorigenin content, the combination of 0.5 mgfL IAAJ 0 mg/L Kinetin provided the highest tectorigenin content of 37.6 4.9 mg/ kg D.W.
(Table 4). It appears that adding kinetin to the culture medium did not result in any increase in the tectorigenin content.
Table 4. Effects of IAA and Kinetin on dry weight, and tectorigenin content in the rhizome tissue of Neomarica gracilis.
__________________________________ _____________________________________________
Plant growth regulators (mg/L) IA.A Kinetin Dry weight (g)' Tectorigenin (mgI kg D.W.)' 0 0 1.08 0.16 bc2 3.0 0.24 d2 0.5 0.1 0. 93 0.06 c 37.0 1.2 a 0.1 0 1.26 0.22 b 11.8 8.4 b 0.5 0 1.02 0.11 bc 37.6 4.9 a 0 1.70 0.08 a 29.0 0.29 c I. Data were collected after 8 weeks culture, and values are means of 3 replicates S.E.
2. Means within a column followed by the same (a to d) letters are not significantly different by Duncan's multiple range test (P>O.05).
Increased tectorigenin content was also found in all tissues cultured in the presence of 2,4-D and NAA, as well as in some tissues cultured with NAA only (Table 5). The highest tectorigenin contents were found in tissues cultured in the presence of 1.0 mg(L NAA and 0.1.mgfL 2,4-D (60.9 0.67 mg/kg D.W.), 0.5 mg/L NAA only (58.9 0.23 mg/kg D.W.), and 0.1 mgfL NAA only (55.5 0.67 mg/kg D.W.). When NAA is used alone, an increase of the NAA concentration led to decreased dry weight. The highest dry weight was obtained with a NAA concentration of 0.1 mgfL (Table 5).
Table 5. Effects of NAA and 2,4-D on dry weight and tectorigenin content in rhizome tissue of Neomarica gracilis.
Plant growth regulators (mgi!) _________________________ 2,4-D NAA Dry weight (g)1 Tectorigenin (mgI kg D.W.)' o 0 1.17 0.02ab2 8.2 0.42 e2 0 0.1 1.39 0.32 a 55.5 0.67 ab 0 0.5 1.03 0.31 bc 58.9 0.23 ab 0 1.0 1.05 0.20 bc 49.0 0.81 bc 0 2.0 0.75 0.02 cd 9.4 0.22 e 0.1 0 0.92 0.l5bc 38.9 1.17 cd 0.1 0.1 1.06 0.03 bc 49.5 1.21 bc 1.
0.1 0.5 0.92 0.13 bc 40.1 0.55 cd 0.1 1.0 0.95 0.01 bc 60.9 0.67 a 0.1 2.0 0.93 0.02 bc 14.5 0.67 e 1.0 0 1.17 0.21 ab 36.7 0.33 cd 1.0 0.1 1.39 0.54 a 35.3 0.23 d 1.0 0.5 1.03 0.12 bc 33.4 0.16 d 1.0 1.0 1.05 0.IObc 16.5 0.80 e 1.0 2.0 0.75 0.03 cd 38.0 1.13 cd 1. Data were collected after 8 weeks culture, and values are means of 3 replicates S.E.
2. Means within a column followed by the same (a to e) letters are not significantly different by Duncan's multiple range test (P>O.05).
Example 4: Growth rate of N. graciis and the tectorigenin content during growth Rhizome tissue was inoculated at an inoculum weight of 3% (g F.W. / 50 ml) in culture medium containing MS basic medium supplemented with 0.1 mg/L NAA or a mixture of I mg/L NAA and 1.0 mgfl 2,4-D. The tissues were weighted and analyzed for tectorigenin content every week for eight consecutive weeks.
As shown in Figure 4, rhizome tissue cultured in an MS basic medium supplemented with 0.1 rng/L NAA grew rapidly during the periods of weeks 3-4 and weeks 6-7. There was little growth during week 5 and week 8. Rhizome tissue cultured in the MS basic medium supplemented with 1 mg/I NAA and 1.0 mg/I 2,4-D showed a similar growth curve.
With regard to the tectorigenin content during growth, the highest content was detected during week 1 under both culture conditions (56 2.82 mg(kg D.W. and 43 2.15 mg/kg D.W. for 0.1 mg/L NAA and 1 mg/L NAAJI.0 mgfL 2,4-D, respectively).
The tectorigenin content reduced significantly on week 2, reaching 7 0.42 mg/kg D.W. at the Lowest point, and gradually increased over weeks 3-4. The tectongenin content then showed another significant reduction at week 5 and increased again during weeks 6-8 (Figure 5).
The total flavonoid content in tissues cultured in MSO (i.e., MS medium without PGR) in solid culture is generally higher than those in MS in liquid culture medium and in TIS for the same amount of time in culture. The total flavonoid content in solid tissue culture containing MSO for about 8 weeks was equivalent to about 10.74 0.31 of -tectorigenin mg/g of dry weight, which is similar to the total flavonoid content extracted from the herb of Belamcanda chinensis, which is well-known for its high content of flavonoid. The total flavonoid content in B. chinensis was about 11.50 0.1 of -tectorigenin mg/g of dry weight based on the same measurement method as used in here.
Example 5: The Effect of ancyinidol on sprout formation and tectorigenin content of cultured N. graciis rhizome tissues Liquid cultured N. gracilis rhizome tissue was inoculated at an inoculum weight of 3% (g F.WJ 100 ml) to a subculture medium containing MS, 0.5 mgIL BA, 0.1 mgIL 2, 4-D and 0.84 mg/L NAA. At day 7 and 14 of the culture, stenlely filtered (0.2 urn) ancymidol was added to the culture medium to a final concentration of 0.5, 1.0, or 2.0 mg/L. The growth of the tissue was monitored and the tectorigenin content analyzed. As shown in Table 6, adding 0.5 or 2.0 mg/L ancymidol to the tissue culture on day 14 resulted in reduced tectorigenin content. Adding ancymidol to the tissue culture on day 7 did not affect the tectorigenin content. Morphologically, however, adding ancymidol to the tissue culture on day 7 led to significant reduction of sprout formation (Figure 6).
Table 6. Effect of ancymidol on the tectorigenin content of N. gracilzs rhizome tissue Ancymidol (mg/L) Adding time Tectorigenin' (day) (mgI kg D.W.) Control 32.81 0.72 ab2 0.5 7 28.31 0.11 b 1.0 7 27.74 0.08 b 2.0 7 35.65 0.18 a 0.5 14 18.59 0.07 c 1.0 14 30.81 0.35 a 2.0 14 13.36 0.13d 1. Data were collected after 3 weeks of culture. Values are means of 3 replicates S.E.
2. Means within a column followed by the same (a to d) letters are not significantly different by Duncan's multiple range test (P>O.05).
Example 6: Effect of inoculum to biomass increase of N. gradiis The TIS culture chamber was divided into four areas and liquid cultured N. gracilis rhizome tissue was inoculated at an inoculuin weight of 1.5, 3, 6 and 9g in each area. The culture medium was MS medium supplemented with PGR as described in samples T12, T17 and T18 of Table 3. The cultured tissue was weighted every ten days. As shown in Figure 7, the inoculum weight of 1.5 g in MS medium supplemented with 1 mg/L kinetin provided the best tissue growth --reaching a fresh weight (6.76 0.33 g) that was 4.5 fold of the inoculum weight in 30 days. All other inoculum weights/PGR combinations led to slower growth (Figuié 7).
To determine the effect of PGRs on the tectorigenin content, ten pieces of liquid cultured N. gracilis rhizome tissue (1.5 g each) were inoculated in culture media containing MS and PGRs described in samples MSO, T13, T14, T15 and T16 of Table 3.
The tissues were harvested on week 4 and analyzed for tectorigenin content. As shown in Table 7, the highest tectorigenin content (48 0.22 mg/kg D.W.) was obtained with MS medium supplemented with 0.1 mg/L 2,4-D and 1.0 mgfL NAA.
Table 7. Effect of PRGs on the growth and tectorigenin content of N. gracilis rhizome tissue in TIS culture Tectorigenin PGRs (mg/i) F.W.(g) Growth rate' (mgI kg D.W.) Control 42.63 3.51 1.84 0.23 22d0.11 Kinetin 1.0 46.12 2.18 2.07 0.15 10 0.15 NA.A 0.1 40.65 2.09 1.71 0.14 38 0.57 NAAO.5 31.47 3.12 1.10 0.21 33 0.38 NAA 1.0 + 2,4-D 0.1 30.84 1.13 1.06 0.08 48 0.22 0.5 BA+0.1 2,4-D+ 0.84 NAA 33.50 1.03 1. 23 0.07 40 0.52 Values are means of 3 replications S.E.
1. Growth rate (Final F.W.-Initial F.W.)Ilnitial F.W.
Example 7: Comparislon of tectorigenin content in N.gradiis rhizome tissues cultured in flask and in TIS Based on the result of Example 2, the combination of 2,4-D and NAA provides higher tectorigenin content than the combination of 1AA and Kinetin. The highest tectorigenin contents were obtained by MS medium supplemented with 0.1 mg/L NAA (tectorigenin 55.5 0.67 mg/ kg D.W.) 0.5 mg/L NAA (tectorigenin 59.9 0.23 mg/ kg D.W.) or 0.1 mg/L NAA and I mgfL 2,4-D (tectorigenin 60.9 0.67 mg/ kg D.W.) The total flavonoids contents were also determined under these culture conditions.
Example 8: Comparision of tectorigenin contents in cultured and wild N. graciis rhizome tissues Table 8 shows the tectorigenin content in cultured and wild N. grad/is rhizome tissues. The results show that wild N.gracilis rhizome tissues had no tectorigenin but the cultured Ngraci/is rhizome tissues had a tectorigenin content of 55.5 0.67 mg/kg D.W.
Table 8. The tectorigenin content of cultured and wild N. grad/is rhizome tissues Tectorigenin (mg/kg D.W.)2 Flask Cultured Rhizome' 55.5 0.67 Wild Rhizome ND3 1. The rhizome culture medium was MS with 0.1 mgI NAA.
2. Values are means of 3 replicates S.E.
3. ND = not detectable Example 9: Extract from cultured N. graciis rhizome tissue inhibits tumor cell growth Extract of the cultured N.gracilis rhizome tissue, obtained using the procedure described in Materials and Methods, was tested in vitro in cultured tumor cells. The extract showed no toxicity to human normal intestine cells in the concentration range of 0.1% to 20%. The extract, however, inhibits the growth of mouse melanoma cells (816-FO) at concentrations of 3.13%, 25% and 50%. The extract was also tested on acute myeloid leukemia cell line PLB985 and human breast cancer cell line MCF7 using lucigenin analysis to monitor the formation of peroxide, followed with analysis of freeradicals by an anti-oxidant analyzer. The result indicated that the extract inhibited the growth of the two tumor cell lines.
The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this specification should be considered as limiting the scope of the present invention. The above-described embodiments of the invention may be modified or varied, and elements added or omitted, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.
Claims (48)
- What is claimed is: 1. An in vitro flavonoid-nch tissue from Neomaricagracilis obtained from a tissue culture preparation which alters the flavonoid content of said N. gracilis, wherein said flavonoids-rich tissue comprises tectorigenin.
- 2. The in vitro flavonoid-rich tissue from N gracilis according to claim 1, wherein said in vitro flavonoids-rich tissue is a rhizome tissue cultured from an N. gracilis tissue capable of proliferating.
- 3. The in vitro flavonoid-rich tissue from N. gracilis according to claim 2, wherein said N. gracilis tissue comprises root, rhizome, leaf, and a basal portion of leaf.
- 4. The in vitro flavonoid-rich tissue from N grad/is according to claim 1, wherein said tissue culture preparation comprises a culture medium which contains a plant growth regulator.
- 5. The in vitro flavonoid-rich tissue from N. gracilis according to claim 4, wherein said plant growth regulator comprises cytokinins or auxins.
- 6. The in vitro flavonoid-rich tissue from N. gracilis according to claim 5, wherein said plant growth regulators are at least one selected from the group consisting of indole-3-acetic acid, 2-4-dichiorophenoxyacetic acid, a-naphthaleneacetic acid, 6-benzyl-aminopurine, and kinetin.
- 7. The in vitro flavonoid-rich tissue from N. grad/is according to claim 4, wherein said plant growth regulator is at a concentration of about 0.01 to 2.0 mgfL.
- 8. The in vitro flavonoid-rich tissue from N. gracilis according to claim 4, wherein said culture medium further comprises a Murashige and Skoog basic salt medium (MS medium).
- 9. The in vitro flavonoid-rich tissue from N. gracilis according to claim 8, wherein said MS medium comprises sodium, potassium, nitrate, ammonium, magnesium, sulfate, calcium, iron, chloride, phosphate, manganese, iodine, borate, zinc, copper, molybdenum, cobalt, or a mixture thereof.
- 10. The in vitro flavonoid-rich tissue from N. gracilis according to claim 4, wherein said culture medium further comprises a carbohydrate.
- 11. The in vitro flavono id-rich tissue from N. gracilis according to claim 10, wherein said carbohydrate is myo-inositol or sucrose or a mixture thereof.
- 12. The in vitro flavonoid-nch tissue from N. gracilis according to claim 4, wherein said culture medium further comprises a vitamin.
- 13. The in vitro flavonoid-rich tissue from N. gracilis according to claim 12, wherein said vitamin is at least one selected from the group consisting of thiamine HCI, pyridoxine HCI, and nicotinic acid.
- 14. The in vitro flavonoid-rich tissue from N. gracilis according to claim 4, wherein said culture medium further comprises an ancymidol.
- 15. The in vitro flavonoid-rich tissue from N. gracilis according to claim 4, wherein said culture medium has a pH of about 5 to 7.
- 16. The in vitro flavonoid-rich tissue from N. gracilis according to claim 1, wherein said tissue culture preparation is a flask culture, a Temporary Immersion System (TIS), or a combination thereof.
- 17. The in vitro flavonoid-rich tissue from N. gracilis according to claim 1, wherein said tectorigenin is in the amount of about 2.5 to 65 mg per Kg of dry tissue weight.
- 18. A method for obtaining said in vitro flavonoid-rich tissue from N. gracilis according to claim 1, comprising: inoculating an N. gracilis tissue in a culture medium of said tissue culture preparation; wherein said N. gracilis tissue is capable of proliferating; and growing said N. gracilis tissue in said tissue culture preparation for a sufficient amount of time to allow a rhizome tissue to form.
- 19. The method according to claim 18, wherein said culture medium is maintained at about 20 C to 30 C.
- 20. The method according to claim 18, wherein said N. gradiis tissue comprises a root, a leaf, a basal portion of a leaf, or a rhizome.
- 21. The method according to claim 18, wherein said tissue culture preparation is a flask culture, a temporary immersion system (TIS), or a combination thereof.
- 22. The method according to claim 18, wherein sufficient amount of time is about 4 to 8 weeks.
- 23. The method according to claim 21, wherein said uS allows said N. gracilis tissue to be immersed in said culture medium for about 1-3 minutes in about every 2-4 hours.
- 24. The method according to claim 18, wherein said culture medium comprises a plant growth regulator, a salt medium, and a carbohydrate.
- 25. The method according to claim 24, wherein said plant growth regulator comprises cytokinins or auxins.
- 26. The method according to claim 24, wherein said plant growth regulator is at least one selected from the group consisting of indole-3-acetic acid, 2-4-dichlorophenoxyacetic acid, a-naphthaleneacetic acid, 6-benzyl-aminopurine, and kinetin.
- 27. The method according to claim 24, wherein said plant growth regulator is at a concentration of about 0.01 to 2.0 mgfL.
- 28. The method according to claim 24, wherein said salt medium is a Murashige and Skoog basic salt medium (MS medium) which comprises sodium, potassium, nitrate, ammonium, magnesium, sulfate, calcium, iron, chloride, phosphate, manganese, iodine, borate, zinc, copper, molybdenum, cobalt, or a mixture thereof.
- 29. The method according to claim 24, wherein said carbohydrate is myo-inositol or sucrose or a mixture thereof.
- 30. The method according to claim 24, wherein said culture medium further comprises a vitamin.
- 31. The in vitro flavonoid-rich tissue from N. graciis according to claim 30, wherein said vitamin is at least one selected from the group consisting of thiamine HCI, pyridoxine HCI, and nicotinic acid.
- 32. The method according to claim 24, wherein said culture medium has a pH of about 5 to 7.
- 33. A method for extracting tectorigenin from said in vitro flavonoid-rich tissue of N. gracilis according to claim 1, comprising: drying said in vitro flavonoid-rich tissue of N. gracilis to obtain a dried flavonoid-rich tissue; adding an alcohol to said dried in vitro flavonoid-nch tissue to form a suspension; heating said suspension to form a heated suspension; and filtering said heated suspension after said heated suspension has cooled off to collect an filtrate which contains said tectorigenin.
- 34. The method according to claim 33, wherein said dried in vitro flavonoid-rich tissue is obtained by subjecting said in vitro flavonoid-rich tissue to freeze-drying.
- 35. The method according to claim 33, wherein said suspension is heated at about 50-70 C.
- 36. The method according to claim 33, wherein said suspension is heated with vibration.
- 37. The method according to claim 36, wherein said vibration is generated by an ultrasonic wave.
- 38. The method according to claim 33, wherein said alcohol is methanol or ethanol.
- 39. The method according to claim 33, wherein said filtrate is collected by passing said heated suspension through a Whatmant No. I filter.
- 40. A method for determining the amount of said tectorigenin in said in vitro flavonoid-rich tissue of N. gracilis according to claim 33, comprising: determining the amount of said tectorigenin by a high performance liquid chromatography (HPLC).
- 41. The method according to ciaim 40, wherein said HPLC contains a Cosmosil 5 C18-AR-II column.
- 42. The method according to claim 40, wherein said tectorigenin is eluted with an eluting solution containing methanol and water (with 0.1% acetic acid) in a volume ratio of 55: 45.
- 43. The method according to claim 40, wherein the amount of said tectorigenin is measured at about 265 nm wavelength using an 1'-tectorigenin as a standard.
- 44. An extract of said in vitro flavonoid-rich tissue of N. gracilis according to claim 33, wherein said extract has anti-tumor activity.
- 45. A flavenoid-rich tissue substantially as hereinbefore described with reference to the accompanying drawings.
- 46. A method for obtaining flavenoid-rich tissue substantially as hereinbefore described with reference to the accompanying drawings.
- 47. A method of extracting tectorigenin substantially as hereinbefore described with reference to the accompanying drawings.
- 48. A method for determining the amount of tectorigenin substantially as described with reference to the accompanying drawings.49 An extract of flavenoid-rich tissue substantially as hereinbefore described with reference to the accompanying drawings. çL
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW095149939A TWI323641B (en) | 2006-12-29 | 2006-12-29 | Tissues with rich flavonoids of neomarica gracilis and belamcanda chinensis and culture methods for the same |
| US11/797,145 US7824914B2 (en) | 2006-12-29 | 2007-05-01 | Flavonoids-rich tissues from Neomarica gracilis and methods for culturing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB0715201D0 GB0715201D0 (en) | 2007-09-12 |
| GB2445213A true GB2445213A (en) | 2008-07-02 |
Family
ID=38529292
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB0715201A Withdrawn GB2445213A (en) | 2006-12-29 | 2007-08-02 | Flavonoid-rich tissue from Neomarica gracilis |
Country Status (2)
| Country | Link |
|---|---|
| DE (1) | DE102007054065A1 (en) |
| GB (1) | GB2445213A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014049546A3 (en) * | 2012-09-27 | 2014-06-26 | Institut National De La Recherche Agronomique | Compositions and methods for enhancing plant growth and development |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040176310A1 (en) * | 2001-05-15 | 2004-09-09 | Wolfgang Wuttke | Use of extracts and preparations from iris plants and tectorigenin as medicaments |
-
2007
- 2007-08-02 GB GB0715201A patent/GB2445213A/en not_active Withdrawn
- 2007-11-13 DE DE102007054065A patent/DE102007054065A1/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040176310A1 (en) * | 2001-05-15 | 2004-09-09 | Wolfgang Wuttke | Use of extracts and preparations from iris plants and tectorigenin as medicaments |
Non-Patent Citations (3)
| Title |
|---|
| Arch. Pharm. Res. Vol.27, 2004, Singab, A.N.B., "Flavonoids from Iris spuria...", pp.1023-1028 * |
| J. Integrative Plant Biol., Vol.47, 2005, Qin, M.-J. et al., "A new isoflavonoid...", pp.1404-1408 * |
| The rhizome tissue multiplacation (sic), tectorigenin and total flavonoid analysis of Neomarica gracilis and Belamcanda chinensis by liquid culture. Available from: http://ethesys.library.ttu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0829106-113658 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014049546A3 (en) * | 2012-09-27 | 2014-06-26 | Institut National De La Recherche Agronomique | Compositions and methods for enhancing plant growth and development |
Also Published As
| Publication number | Publication date |
|---|---|
| GB0715201D0 (en) | 2007-09-12 |
| DE102007054065A1 (en) | 2008-07-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7824914B2 (en) | Flavonoids-rich tissues from Neomarica gracilis and methods for culturing the same | |
| Abdoli et al. | Morphological, physiological, cytological and phytochemical studies in diploid and colchicine-induced tetraploid plants of Echinacea purpurea (L.) | |
| Majdi et al. | Induction of tetraploidy to feverfew (Tanacetum parthenium Schulz-Bip.): Morphological, physiological, cytological, and phytochemical changes | |
| Lata et al. | TDZ-induced high frequency plant regeneration through direct shoot organogenesis in Stevia rebaudiana Bertoni: an important medicinal plant and a natural sweetener | |
| US20080311634A1 (en) | Flavonoids-rich tissue from Belamcanda chinensis and methods for culturing the same | |
| Shohael et al. | Application of bioreactor system for large-scale production of Eleutherococcus sessiliflorus somatic embryos in an air-lift bioreactor and production of eleutherosides | |
| Maliński et al. | Various in vitro systems of Ragged Robin (Lychnis flos-cuculi L.): a new potential source of phytoecdysteroids? | |
| Liu et al. | Comparison of phytochemical and antioxidant activities in micropropagated and seed-derived Salvia miltiorrhiza plants | |
| Aung et al. | Effects of Different Natural Extracts and Plant Growth Regulators on Plant Regeneration and Callus Induction from Pseudobulbs Explants through in vitro Seed Germination of Endangered Orchid Bulbophyllum auricomum Lindl. | |
| Purse | Phloem exudate of Perilla crispa and its effects on flowering of P. crispa shoot explants | |
| Tsay et al. | Tissue culture technology of Chinese medicinal plant resources in Taiwan and their sustainable utilization | |
| WO2007107803A2 (en) | Process and modified media for preparing callus- and cell suspension cultures of hypericum perforatum l. | |
| Cheng et al. | Somatic embryogenesis and triterpenoid saponin production in Aralia elata (Miq.) Seem | |
| Ionkova | Optimization of flavonoid production in cell cultures of Astragalus missouriensis Nutt.(Fabaceae) | |
| GB2445213A (en) | Flavonoid-rich tissue from Neomarica gracilis | |
| D'Angiolillo et al. | In vitro cultures of Bituminaria bituminosa: pterocarpan, furanocoumarin and isoflavone production and cytotoxic activity evaluation | |
| Karakaş et al. | Determination of hypericin content in callus and cell suspension cultures of Hypericum triquetrifolium Turra | |
| Uikey et al. | Secondary metabolite production of reserpine and ajmalicine in Rauvolfia serpentina (L.) Benth. through callus and cell suspension culture | |
| Siddiqui et al. | Comparative analysis of hyoscine in wild-type and in vitro grown datura innoxia by high performance liquid chromatography | |
| Daffalla et al. | Secondary metabolites accumulation and production through in vitro cultures | |
| KR20200042701A (en) | Production method for cultured root of leguminous plants comprising high-content of coumestrol | |
| Faramayuda et al. | ANALYSIS OF SECONDARY METABOLITES OF CALLUS OF RAMBUTAN Nephelium lappaceum L | |
| Taha et al. | In vitro production of caffic acid derivatives in calli and regenerate cultures of Echinacea angustifolia Dc and Echinacea Pallida Nutt | |
| CN117866873A (en) | Method for establishing mugwort suspension cell line | |
| Nyembo et al. | Establishment of callus derived from Jatropha curcas L. petiole explants and phytochemical screening |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |