CN100362012C - Method for synthesizing secoiridoid glycosides compound - Google Patents
Method for synthesizing secoiridoid glycosides compound Download PDFInfo
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- CN100362012C CN100362012C CNB031261116A CN03126111A CN100362012C CN 100362012 C CN100362012 C CN 100362012C CN B031261116 A CNB031261116 A CN B031261116A CN 03126111 A CN03126111 A CN 03126111A CN 100362012 C CN100362012 C CN 100362012C
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- 238000000034 method Methods 0.000 title claims abstract description 29
- 229930182470 glycoside Natural products 0.000 title claims abstract description 18
- -1 glycosides compound Chemical class 0.000 title claims abstract description 18
- 230000002194 synthesizing effect Effects 0.000 title abstract 2
- 239000007787 solid Substances 0.000 claims abstract description 28
- 238000000605 extraction Methods 0.000 claims abstract description 17
- 230000006698 induction Effects 0.000 claims abstract description 5
- 238000009630 liquid culture Methods 0.000 claims abstract description 4
- 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 20
- 229930006000 Sucrose Natural products 0.000 claims description 20
- 239000003960 organic solvent Substances 0.000 claims description 20
- 239000005720 sucrose Substances 0.000 claims description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 18
- 235000015097 nutrients Nutrition 0.000 claims description 17
- 230000001954 sterilising effect Effects 0.000 claims description 17
- 238000004659 sterilization and disinfection Methods 0.000 claims description 16
- 229920001817 Agar Polymers 0.000 claims description 15
- 239000008272 agar Substances 0.000 claims description 15
- 238000005303 weighing Methods 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 14
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 12
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 11
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 10
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims description 10
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- 150000001298 alcohols Chemical class 0.000 claims description 8
- 239000012533 medium component Substances 0.000 claims description 8
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 7
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 claims description 7
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 7
- 239000011591 potassium Substances 0.000 claims description 7
- 229910052700 potassium Inorganic materials 0.000 claims description 7
- 235000019157 thiamine Nutrition 0.000 claims description 7
- 150000003544 thiamines Chemical class 0.000 claims description 7
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 6
- 239000004327 boric acid Substances 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 6
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 6
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 6
- 238000004090 dissolution Methods 0.000 claims description 6
- 239000011790 ferrous sulphate Substances 0.000 claims description 6
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 6
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 6
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- 229960004839 potassium iodide Drugs 0.000 claims description 6
- 235000007715 potassium iodide Nutrition 0.000 claims description 6
- 239000004323 potassium nitrate Substances 0.000 claims description 6
- 235000010333 potassium nitrate Nutrition 0.000 claims description 6
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 6
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical class [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 claims description 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- 229960001763 zinc sulfate Drugs 0.000 claims description 6
- LNETULKMXZVUST-UHFFFAOYSA-N 1-naphthoic acid Chemical compound C1=CC=C2C(C(=O)O)=CC=CC2=C1 LNETULKMXZVUST-UHFFFAOYSA-N 0.000 claims description 5
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 5
- 239000005972 6-Benzyladenine Substances 0.000 claims description 5
- 241000196324 Embryophyta Species 0.000 claims description 5
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 5
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 5
- 235000019253 formic acid Nutrition 0.000 claims description 5
- 239000003617 indole-3-acetic acid Substances 0.000 claims description 5
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound 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 5
- 229960000367 inositol Drugs 0.000 claims description 5
- 229960003512 nicotinic acid Drugs 0.000 claims description 5
- 235000001968 nicotinic acid Nutrition 0.000 claims description 5
- 239000011664 nicotinic acid Substances 0.000 claims description 5
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 claims description 5
- 235000008160 pyridoxine Nutrition 0.000 claims description 5
- 239000011677 pyridoxine Substances 0.000 claims description 5
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- 241000735234 Ligustrum Species 0.000 claims description 4
- 241000207834 Oleaceae Species 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 4
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 4
- 150000002576 ketones Chemical class 0.000 claims description 4
- 229940045641 monobasic sodium phosphate Drugs 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 4
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 claims description 3
- 230000009514 concussion Effects 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000010992 reflux Methods 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 238000000638 solvent extraction Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 150000002894 organic compounds Chemical class 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 abstract 4
- 238000006243 chemical reaction Methods 0.000 abstract 1
- 238000011177 media preparation Methods 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 26
- RFWGABANNQMHMZ-UHFFFAOYSA-N 8-acetoxy-7-acetyl-6,7,7a,8-tetrahydro-5H-benzo[g][1,3]dioxolo[4',5':4,5]benzo[1,2,3-de]quinoline Natural products CC=C1C(CC(=O)OCCC=2C=C(O)C(O)=CC=2)C(C(=O)OC)=COC1OC1OC(CO)C(O)C(O)C1O RFWGABANNQMHMZ-UHFFFAOYSA-N 0.000 description 4
- HKVGJQVJNQRJPO-UHFFFAOYSA-N Demethyloleuropein Natural products O1C=C(C(O)=O)C(CC(=O)OCCC=2C=C(O)C(O)=CC=2)C(=CC)C1OC1OC(CO)C(O)C(O)C1O HKVGJQVJNQRJPO-UHFFFAOYSA-N 0.000 description 4
- RFWGABANNQMHMZ-HYYSZPHDSA-N Oleuropein Chemical compound O([C@@H]1OC=C([C@H](C1=CC)CC(=O)OCCC=1C=C(O)C(O)=CC=1)C(=O)OC)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O RFWGABANNQMHMZ-HYYSZPHDSA-N 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 229960004756 ethanol Drugs 0.000 description 4
- RFWGABANNQMHMZ-CARRXEGNSA-N oleuropein Natural products COC(=O)C1=CO[C@@H](O[C@H]2O[C@@H](CO)[C@H](O)[C@@H](O)[C@@H]2O)C(=CC)[C@H]1CC(=O)OCCc3ccc(O)c(O)c3 RFWGABANNQMHMZ-CARRXEGNSA-N 0.000 description 4
- 235000011576 oleuropein Nutrition 0.000 description 4
- 238000003808 methanol extraction Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241000207840 Jasminum Species 0.000 description 2
- 241000795633 Olea <sea slug> Species 0.000 description 2
- 241001104043 Syringa Species 0.000 description 2
- 235000004338 Syringa vulgaris Nutrition 0.000 description 2
- 230000036983 biotransformation Effects 0.000 description 2
- 229960000935 dehydrated alcohol Drugs 0.000 description 2
- 229940064880 inositol 100 mg Drugs 0.000 description 2
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000576449 Fontanesia Species 0.000 description 1
- 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 description 1
- 241000830535 Ligustrum lucidum Species 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 241000333181 Osmanthus Species 0.000 description 1
- 235000019082 Osmanthus Nutrition 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229910052796 boron Inorganic materials 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 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 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
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- 238000010189 synthetic method Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
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- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
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- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
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Abstract
The present invention relates to a method for synthesizing secoiridoid glycosides compounds, which belongs to the technical field of the biolgoical conversion synthesis of organic compounds. The present invention comprises culture medium preparation, explant induction, tissue (cell) cultivation and target molecule extraction. The present invention can only utilize a solid culture medium to make cultivation and extraction, can also utilize the solid culture medium to make the cultivation and then utilize a liquid culture medium to make the cultivation and the extraction. The present invention has the advantages of simple and feasible process, high product yield, low production cost and easy industrial application.
Description
Technical field
The invention belongs to the bio-transformation synthesis technical field of organic compound, the method for the synthetic secoiridoid glycoside compound of particularly a kind of utilization tissue (cell) culture method.
Background technology
That the secoiridoid glycoside compound is that a class has is antibiotic, the material of anti-inflammatory, effect such as antiviral, anti-oxidant, anticancer, hypoglycemic, and toxicity is very low, can be used for medical treatment and nourishing function aspect.But this compounds content in natural phant is very low, and the chemical structure complexity, utilizes chemical process to carry out complete synthesis very difficulty, and uneconomical, has seriously limited its further development and utilization.
Summary of the invention
The object of the present invention is to provide a kind of bio-transformation synthetic method that is suitable for the synthetic secoiridoid glycoside compound of suitability for industrialized production promptly to organize (cell) cultural method.
In order to achieve the above object, the present invention adopts following technical scheme: a kind of method of synthetic secoiridoid glycoside compound, comprise the preparation of substratum, four steps of extraction of inducing, organizing (cell) cultivation and target molecule of explant: in the explant induction step, be inoculated on the solid medium after getting Oleaceae Genus Syringa, Ligustrum, sweet-scented osmanthus genus, Olea, Jasminum, fontanesia Shu He the plant leaf of Ash genus, leaf bud or rhizome sterilization, cultivated 15-108 days in 15-32 ℃; At the target molecule extraction step, place extractor to use organic solvent refluxing extraction 2-6 hour or cold soaking 8-48 hour the callus of gathering in the crops, remove organic solvent and get target molecule secoiridoid glycoside compound.
Contain sal epsom 160-689mg/l in the solid medium, potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC or sum of the two 70-350mg/l, saltpetre 860-4200mg/l, ammonium nitrate 725-4600mg/l, calcium chloride 180-980mg/l, thiamines 0.05-1.25mg/l, inositol 25-250mg/l, boric acid 1.6-26.8mg/l, manganous sulfate 3.9-62.5mg/l, zinc sulfate 2.1-32.2mg/l, Sodium orthomolybdate 0.0125-0.0725mg/l, copper sulfate 0.0125-0.0725mg/l, cobalt chloride 0.0125-0.0725mg/l, potassiumiodide 0.0215-2.65mg/l, ferrous sulfate 6.5-65.5mg/l, EDTA sodium salt 9.8-76.4mg/l or EDTA molysite 21.5-86mg/l, 2,4-D, N
6-furfuryladenine, 6-benzyladenine, one of 6-chaff aminoadenine or more than two kinds and 0.05-25mg/l, naphthylacetic acid, naphthoic acid, indole-3-acetic acid, 4-amino-3,5, one of 6-trichloropyridine formic acid or more than two kinds and 0.1-50mg/l, sucrose 12000-60000mg/l, agar 4500-32000mg/l, take by weighing one by one except that sucrose, the medium component that agar is outer, use water dissolution, mixing and thin up are to desired concn, take by weighing formula ratio sucrose and agar then, add in the above-mentioned solution and dissolve, transfer PH=4.5-7.0, after sterilization, get solid medium.
Also contain pyridoxol 0.125-1.25mg/l, nicotinic acid 0.025-0.25mg/l in the solid medium.
Organic solvent comprises alcohols, ketone and ether organic solvent.
Organic solvent is methyl alcohol or the ethanol in the alcohols, and consumption is 10-20 a times of callus weight.
A kind of method of synthetic secoiridoid glycoside compound, comprise the preparation of solid medium and liquid nutrient medium, four steps of extraction of inducing, organizing (cell) cultivation and target product of explant: in the explant induction step, be inoculated on the solid medium after getting Oleaceae Genus Syringa, Ligustrum, Olea, Jasminum He the plant leaf of Ash genus, leaf bud or rhizome sterilization, cultivated 14-49 days in 15-32 ℃; Taking out callus again transfers in the liquid nutrient medium in concussion incubator or shaking table or bio-reactor or reactor and cultivated 10-60 days at 18-32 ℃; At the target product extraction step, culture and substratum filtration are earlier separately placed the extractor organic solvent extraction respectively again, remove organic solvent and get target product secoiridoid glycoside compound.
Contain sal epsom 160-689mg/l in the liquid nutrient medium, potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC or sum of the two 70-350mg/l, saltpetre 860-4200mg/l, ammonium nitrate 725-4600mg/l, calcium chloride 180-980mg/l, thiamines 0.05-1.25mg/l, inositol 25-250mg/l, boric acid 1.6-26.8mg/l, manganous sulfate 3.9-62.5mg/l, zinc sulfate 2.1-32.2mg/l, Sodium orthomolybdate 0.0125-0.0725mg/l, copper sulfate 0.0125-0.0725mg/l, cobalt chloride 0.0125-0.0725mg/l, potassiumiodide 0.0215-2.65mg/l, ferrous sulfate 6.5-65.5mg/l, EDTA sodium salt 9.8-76.4mg/l or EDTA molysite 21.5-86mg/l, 2,4-D, N
6One of-furfuryladenine, 6-benzyladenine, 6-chaff aminoadenine or more than two kinds and 0.05-25mg/l, naphthylacetic acid, naphthoic acid, indole-3-acetic acid, 4-amino-3,5, one of 6-trichloropyridine formic acid or more than two kinds and 0.1-50mg/l, sucrose 12000-60000mg/l, take by weighing the medium component except that sucrose one by one, with water dissolution, mix and thin up to desired concn, take by weighing formula ratio sucrose then, add in the above-mentioned solution and dissolve, transfer PH=4.5-7.0, after sterilization, get liquid nutrient medium; Solid medium is than increasing the agar composition in the liquid culture based formulas, content is 4500-19500mg/l, its method for making is: take by weighing the medium component except that sucrose, agar, with water dissolution, mix and thin up to desired concn, take by weighing formula ratio sucrose and agar then, add in the above-mentioned solution and dissolve, transfer PH=4.5-7.0, after sterilization, get solid medium.
Respectively contain pyridoxol 0.125-1.25mg/l, nicotinic acid 0.025-0.25mg/l in solid medium and the liquid nutrient medium.
Organic solvent comprises alcohols, ketone and ether organic solvent.
Organic solvent is methyl alcohol or the ethanol in the alcohols, and consumption is 10-20 a times of callus.
The present invention adopts the synthetic secoiridoid glycoside compound of tissue (cell) cultural method in biology and the cell engineering; not limited by natural resources; and help preserving the ecological environment; the plant cell culture medium of substratum for extensively adopting in tissue (cell) cultivation in the method; composition is easy to get; contain macroelement such as nitrogen, phosphorus, potassium in the substratum; trace elements such as boron, zinc, manganese, molybdenum, copper, cobalt, sodium, iodine, iron; VITAMIN and 2 such as thiamines, pyridoxol, nicotinic acid, inositol, 4-D, N
6-furfuryladenine, 6-benzyladenine, 6-chaff aminoadenine, naphthylacetic acid, naphthoic acid, indole-3-acetic acid, 4-amino-3,5, plant growth regulating substances such as 6-trichloropyridine formic acid, can provide tissue (cell) to cultivate required various nutritive ingredients, the culture condition gentleness, easy extraction.Entire method is easy to suitability for industrialized production.In two kinds of methods of the present invention, first method only adopts solid medium to cultivate; Second method adopts the solid medium cultivation to change liquid nutrient medium then over to earlier and cultivates, and is easier to large-scale industrialization production, and shortens incubation time, and product yield improves greatly, and production cost is reduced greatly.
Description of drawings
Fig. 1 is the chemical structural formula of target product secoiridoid glycoside compound representative oleuropein among the present invention.
Embodiment
Embodiment 1, and a kind of method of synthetic secoiridoid glycoside compound is got sal epsom 370mg, potassium primary phosphate 170mg, saltpetre 1900mg, ammonium nitrate 1650mg, calcium chloride 440mg, thiamines 1.0mg, inositol 100mg, boric acid 6.2mg, manganous sulfate 15.6mg, zinc sulfate 8.6mg, Sodium orthomolybdate 0.025mg, copper sulfate 0.025mg, cobalt chloride 0.025mg, potassiumiodide 0.085mg, ferrous sulfate 27.8mg, EDTA sodium salt 37.3mg, 2,4-D1.0mg, 6-chaff aminoadenine 2.0mg, naphthylacetic acid 3.0mg, sucrose 30000mg, agar 12000mg takes by weighing one by one except that sucrose, the medium component that agar is outer is used deionized water dissolving, mix, take by weighing formula ratio sucrose and agar then, add to dissolve in the above-mentioned solution and add deionized water and be diluted to 1000ml, transfer PH=5.8, through 121 ℃ of sterilizations after 25 minutes solid medium.
On the solid medium that is inoculated in after daphne lilac blade or young shoot or the sterilization of tender stem after the sterilization, in 25 ℃ of incubators, induce and produce callus and cultivated 49 days, take out product, place apparatus,Soxhlet's to add the dehydrated alcohol extraction 2 times of 10 times of weight at every turn, steam ethanol then, get target molecule.Its representative oleuropein structural formula is seen Fig. 1, and its NMR data are listed in the table 1.
Embodiment 2, and the glossy privet blade after will sterilizing in the present embodiment places on the solid medium after the sterilization, induce in 28 ℃ of incubators and cultivate 49 days, take out product, place apparatus,Soxhlet's to add the dehydrated alcohol extraction 2 times of 15 times of weight at every turn, steam ethanol then, target molecule.Other is with embodiment 1.
Insert on the solid medium after getting the sterilization of daphne lilac blade, cultivated 42 days for 28 ℃, the callus that obtains is changed in the liquid nutrient medium, in the shaking culture case, cultivated 21 days for 25 ℃, filter separately culture and nutrient solution.Culture adds the methyl alcohol of 10 times of weight with methanol extraction 2 times at every turn; Nutrient solution adds the methyl alcohol of 3 times of weight with methanol extraction 2 times at every turn.The united extraction thing is a target product.Its representative oleuropein structural formula is seen Fig. 1, and its NMR data are listed in the table 1.
NMR data (the CD of table 1 oleuropein
3COCD
3)
H | δ H(ppm) | J(Hz) | C | δ C(ppm) |
1 | 5.91s | 1 | 94.3 | |
3 | 7.47 |
3 | 154.1 | |
5 | 3.94dd | 9.6,4.0 | 4 | 108.7 |
6a | 2.39dd | 14.0,9.6 | 5 | 31.1 |
6b | 2.70dd | 14.0,4.0 | 6 | 40.4 |
8 | 6.01q | 6.8 | 7 | 171.6 |
10 | 1.64d | 6.8 | 8 9 10 11 | 124.0 129.8 13.3 167.1 |
OMe | 3.67s | OMe | 51.4 |
1′ | 4.87d | 8.0 | 1′ | 100.1 |
2′,4′,5′ | 3.41~3.45m | 2′ | 74.1 | |
3′ | 3.55t | 8.8 | 3′ | 77.2 |
6′a | 3.66dd | 11.6,6.0 | 4′ | 70.9 |
6′b | 3.87d | 11.6 | 5′ 6′ | 77.4 62.3 |
1″a | 4.07dt | 10.8,7.2 | 1″ | 65.9 |
1″b | 4.18dt | 10.8,7.2 | 2″ | 34.6 |
2″ | 2.74t | 7.2 | 3″ | 130.0 |
4″ | 6.73d | 1.6 | 4″ | 116.5 |
7″ | 6.74d | 8.0 | 5″ | 145.4 |
8″ | 6.56dd | 8.0,1.6 | 6″ 7″ 8″ | 144.1 115.8 120.7 |
Claims (10)
1. the method for a synthetic secoiridoid glycoside compound is characterized in that, comprises four steps of extraction of the inducing of preparation, explant, tissue culture and the target molecule of substratum; In the explant induction step, be inoculated on the solid medium after getting the plant leaf, leaf bud of Oleaceae Genus Syringa, Ligustrum or rhizome sterilization, cultivated 15-108 days in 15-32 ℃; At the target molecule extraction step, place extractor to use organic solvent refluxing extraction 2-6 hour or cold soaking 8--48 hour the callus of gathering in the crops, remove organic solvent and get target molecule secoiridoid glycoside compound.
2. the method for claim 1, it is characterized in that, contain sal epsom 160-689mg/l in the solid medium, potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC or sum of the two 70-350mg/l, saltpetre 860-4200mg/l, ammonium nitrate 725-4600mg/l, calcium chloride 180-980mg/l, thiamines 0.05-1.25mg/l, inositol 25-250mg/l, boric acid 1.6-26.8mg/l, manganous sulfate 3.9-62.5mg/l, zinc sulfate 2.1-32.2mg/l, Sodium orthomolybdate 0.0125-0.0725mg/l, copper sulfate 0.0125-0.0725mg/l, cobalt chloride 0.0125-0.0725mg/l, potassiumiodide 0.0215-2.65mg/l, ferrous sulfate 6.5-65.5mg/l, EDTA sodium salt 9.8-76.4mg/l or EDTA molysite 21.5-86mg/l, 2,4-D, N
6-furfuryladenine, 6-benzyladenine, one of 6-chaff aminoadenine or more than two kinds and 0.05-25mg/l, naphthylacetic acid, naphthoic acid, indole-3-acetic acid, 4-amino-3,5, one of 6-trichloropyridine formic acid or more than two kinds and 0.1-50mg/l, sucrose 12000-60000mg/l, agar 4500-32000 mg/l, take by weighing one by one except that sucrose, the medium component that agar is outer, use water dissolution, mixing and thin up are to desired concn, take by weighing formula ratio sucrose and agar then, add in the above-mentioned solution and dissolve, transfer PH=4.5-7.0, after sterilization, get solid medium.
3. method as claimed in claim 2 is characterized in that, also contains pyridoxol 0.125-1.25mg/l, nicotinic acid 0.025-0.25mg/l in the solid medium.
4. as claim 1,2 or 3 described methods, it is characterized in that organic solvent comprises alcohols, ketone and ether organic solvent.
5. method as claimed in claim 4 is characterized in that, organic solvent is methyl alcohol or the ethanol in the alcohols, and consumption is 10-20 a times of callus weight.
6. the method for a synthetic secoiridoid glycoside compound is characterized in that, comprises four steps of extraction of the inducing of preparation, explant, tissue culture and the target product of solid medium and liquid nutrient medium; In the explant induction step, be inoculated on the solid medium after getting the plant leaf, leaf bud of Oleaceae Genus Syringa, Ligustrum or rhizome sterilization, cultivated 14-49 days in 15-32 ℃; Taking out callus again transfers in the liquid nutrient medium in concussion incubator or shaking table or bio-reactor or reactor and cultivated 10-60 days at 18-32 ℃; At the target product extraction step, culture and substratum filtration are earlier separately placed the extractor organic solvent extraction respectively again, remove organic solvent and get target product secoiridoid glycoside compound.
7. method as claimed in claim 6, it is characterized in that, contain sal epsom 160-689mg/l in the liquid nutrient medium, potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC or sum of the two 70-350mg/l, saltpetre 860-4200mg/l, ammonium nitrate 725-4600mg/l, calcium chloride 180-980mg/l, thiamines 0.05-1.25mg/l, inositol 25-250mg/l, boric acid 1.6-26.8mg/l, manganous sulfate 3.9-62.5mg/l, zinc sulfate 2.1-32.2mg/l, Sodium orthomolybdate 0.0125-0.0725mg/l, copper sulfate 0.0125-0.0725mg/l, cobalt chloride 0.0125-0.0725mg/l, potassiumiodide 0.0215-2.65mg/l, ferrous sulfate 6.5-65.5mg/l, EDTA sodium salt 9.8-76.4mg/l or EDTA molysite 21.5-86mg/l, 2,4-D, N
6One of-furfuryladenine, 6-benzyladenine, 6-chaff aminoadenine or more than two kinds and 0.05-25mg/l, naphthylacetic acid, naphthoic acid, indole-3-acetic acid, 4-amino-3,5, one of 6-trichloropyridine formic acid or more than two kinds and 0.1-50mg/l, sucrose 12000-60000mg/l, take by weighing the medium component except that sucrose one by one, with water dissolution, mix and thin up to desired concn, take by weighing formula ratio sucrose then, add in the above-mentioned solution and dissolve, transfer PH=4.5-7.0, after sterilization, get liquid nutrient medium; Solid medium is than increasing the agar composition in the liquid culture based formulas, content is 4500-19500mg/l, its method for making is: take by weighing the medium component except that sucrose, agar, with water dissolution, mix and thin up to desired concn, take by weighing formula ratio sucrose and agar then, add in the above-mentioned solution and dissolve, transfer PH=4.5-7.0, after sterilization, get solid medium.
8. method as claimed in claim 7 is characterized in that, respectively contains pyridoxol 0.125-1.25mg/l, nicotinic acid 0.025-0.25mg/l in solid medium and the liquid nutrient medium.
9. as claim 6,7 or 8 described methods, it is characterized in that organic solvent comprises alcohols, ketone and ether organic solvent.
10. method as claimed in claim 9 is characterized in that, organic solvent is methyl alcohol or the ethanol in the alcohols, and consumption is 10-20 a times of callus.
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JPH0223880A (en) * | 1988-07-13 | 1990-01-26 | Kanebo Ltd | Production of bitter component of plant of family gentianaceae |
US6197308B1 (en) * | 1998-07-23 | 2001-03-06 | Creagri L.L.C. | Water-soluble extract from olives |
JP2002128678A (en) * | 2000-10-17 | 2002-05-09 | Tama Seikagaku Kk | Method for producing extract composition containing oleuropein |
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JPH0223880A (en) * | 1988-07-13 | 1990-01-26 | Kanebo Ltd | Production of bitter component of plant of family gentianaceae |
US6197308B1 (en) * | 1998-07-23 | 2001-03-06 | Creagri L.L.C. | Water-soluble extract from olives |
JP2002128678A (en) * | 2000-10-17 | 2002-05-09 | Tama Seikagaku Kk | Method for producing extract composition containing oleuropein |
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