CN107417479B - Method for separating and enriching allelochemicals of water culture root of camptothecin-containing plants - Google Patents
Method for separating and enriching allelochemicals of water culture root of camptothecin-containing plants Download PDFInfo
- Publication number
- CN107417479B CN107417479B CN201710425639.8A CN201710425639A CN107417479B CN 107417479 B CN107417479 B CN 107417479B CN 201710425639 A CN201710425639 A CN 201710425639A CN 107417479 B CN107417479 B CN 107417479B
- Authority
- CN
- China
- Prior art keywords
- silica gel
- allelochemicals
- water culture
- petroleum ether
- root
- 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.)
- Active
Links
- 239000003627 allelochemical Substances 0.000 title claims abstract description 50
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 26
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 title claims abstract description 23
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 title claims abstract description 23
- 229940127093 camptothecin Drugs 0.000 title claims abstract description 23
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 title claims abstract description 23
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 108
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 96
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 70
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 63
- 239000000741 silica gel Substances 0.000 claims abstract description 61
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 61
- 239000003208 petroleum Substances 0.000 claims abstract description 48
- 241000196324 Embryophyta Species 0.000 claims abstract description 45
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000003480 eluent Substances 0.000 claims abstract description 29
- 210000000416 exudates and transudate Anatomy 0.000 claims abstract description 19
- 241000759905 Camptotheca acuminata Species 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 16
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 claims abstract description 15
- 238000010828 elution Methods 0.000 claims abstract description 13
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 13
- 239000003501 hydroponics Substances 0.000 claims abstract description 12
- 238000004440 column chromatography Methods 0.000 claims abstract description 8
- 238000002390 rotary evaporation Methods 0.000 claims abstract description 6
- 239000000126 substance Substances 0.000 claims abstract description 6
- 230000028327 secretion Effects 0.000 claims description 31
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- 239000000843 powder Substances 0.000 claims description 17
- MGWAVDBGNNKXQV-UHFFFAOYSA-N diisobutyl phthalate Chemical compound CC(C)COC(=O)C1=CC=CC=C1C(=O)OCC(C)C MGWAVDBGNNKXQV-UHFFFAOYSA-N 0.000 claims description 16
- ICKWICRCANNIBI-UHFFFAOYSA-N 2,4-di-tert-butylphenol Chemical compound CC(C)(C)C1=CC=C(O)C(C(C)(C)C)=C1 ICKWICRCANNIBI-UHFFFAOYSA-N 0.000 claims description 14
- DOIRQSBPFJWKBE-UHFFFAOYSA-N dibutyl phthalate Chemical compound CCCCOC(=O)C1=CC=CC=C1C(=O)OCCCC DOIRQSBPFJWKBE-UHFFFAOYSA-N 0.000 claims description 12
- QPFMBZIOSGYJDE-UHFFFAOYSA-N 1,1,2,2-tetrachloroethane Chemical compound ClC(Cl)C(Cl)Cl QPFMBZIOSGYJDE-UHFFFAOYSA-N 0.000 claims description 10
- JLTDJTHDQAWBAV-UHFFFAOYSA-N N,N-dimethylaniline Chemical compound CN(C)C1=CC=CC=C1 JLTDJTHDQAWBAV-UHFFFAOYSA-N 0.000 claims description 10
- 238000004458 analytical method Methods 0.000 claims description 9
- 238000004587 chromatography analysis Methods 0.000 claims description 9
- 238000011068 loading method Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 239000012074 organic phase Substances 0.000 claims description 9
- 239000012043 crude product Substances 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 8
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims description 8
- DJDSLBVSSOQSLW-UHFFFAOYSA-N mono(2-ethylhexyl) phthalate Chemical compound CCCCC(CC)COC(=O)C1=CC=CC=C1C(O)=O DJDSLBVSSOQSLW-UHFFFAOYSA-N 0.000 claims description 8
- DJDSLBVSSOQSLW-LBPRGKRZSA-N phthalic acid mono-2-ethylhexyl ester Natural products CCCC[C@H](CC)COC(=O)C1=CC=CC=C1C(O)=O DJDSLBVSSOQSLW-LBPRGKRZSA-N 0.000 claims description 8
- IWCQVOVBDXJJDF-UHFFFAOYSA-N benzene;chromium;cyclohexane Chemical compound [Cr].[CH-]1[CH-][CH-][CH-][CH-][CH-]1.C1=CC=CC=C1 IWCQVOVBDXJJDF-UHFFFAOYSA-N 0.000 claims description 7
- PGSWEKYNAOWQDF-UHFFFAOYSA-N 3-methylcatechol Chemical compound CC1=CC=CC(O)=C1O PGSWEKYNAOWQDF-UHFFFAOYSA-N 0.000 claims description 6
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims description 6
- FLIACVVOZYBSBS-UHFFFAOYSA-N Methyl palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC FLIACVVOZYBSBS-UHFFFAOYSA-N 0.000 claims description 6
- AFBPFSWMIHJQDM-UHFFFAOYSA-N N-methylaniline Chemical compound CNC1=CC=CC=C1 AFBPFSWMIHJQDM-UHFFFAOYSA-N 0.000 claims description 6
- BJQHLKABXJIVAM-UHFFFAOYSA-N bis(2-ethylhexyl) phthalate Chemical compound CCCCC(CC)COC(=O)C1=CC=CC=C1C(=O)OCC(CC)CCCC BJQHLKABXJIVAM-UHFFFAOYSA-N 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 238000001179 sorption measurement Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- -1 4-oxo-7, 7-dinitro-4, 5,6, 7-tetrahydro-2H-benzotriazole Chemical compound 0.000 claims description 5
- 241000759909 Camptotheca Species 0.000 claims description 5
- 244000025254 Cannabis sativa Species 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 5
- 238000000265 homogenisation Methods 0.000 claims description 5
- 239000012535 impurity Substances 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 239000002975 chemoattractant Substances 0.000 claims description 4
- 238000011049 filling Methods 0.000 claims description 4
- DWANEFRJKWXRSG-UHFFFAOYSA-N 1,2-tetradecanediol Chemical compound CCCCCCCCCCCCC(O)CO DWANEFRJKWXRSG-UHFFFAOYSA-N 0.000 claims description 3
- KGRVJHAUYBGFFP-UHFFFAOYSA-N 2,2'-Methylenebis(4-methyl-6-tert-butylphenol) Chemical compound CC(C)(C)C1=CC(C)=CC(CC=2C(=C(C=C(C)C=2)C(C)(C)C)O)=C1O KGRVJHAUYBGFFP-UHFFFAOYSA-N 0.000 claims description 3
- SIXWIUJQBBANGK-UHFFFAOYSA-N 4-(4-fluorophenyl)-1h-pyrazol-5-amine Chemical compound N1N=CC(C=2C=CC(F)=CC=2)=C1N SIXWIUJQBBANGK-UHFFFAOYSA-N 0.000 claims description 3
- ZYASLTYCYTYKFC-UHFFFAOYSA-N 9-methylidenefluorene Chemical compound C1=CC=C2C(=C)C3=CC=CC=C3C2=C1 ZYASLTYCYTYKFC-UHFFFAOYSA-N 0.000 claims description 3
- QVWDCTQRORVHHT-UHFFFAOYSA-N tropone Chemical compound O=C1C=CC=CC=C1 QVWDCTQRORVHHT-UHFFFAOYSA-N 0.000 claims description 3
- 238000001704 evaporation Methods 0.000 claims description 2
- 238000011010 flushing procedure Methods 0.000 claims description 2
- 238000005374 membrane filtration Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 235000010290 biphenyl Nutrition 0.000 claims 1
- 239000004305 biphenyl Substances 0.000 claims 1
- 229910052804 chromium Inorganic materials 0.000 claims 1
- 239000011651 chromium Substances 0.000 claims 1
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 claims 1
- 238000000926 separation method Methods 0.000 abstract description 22
- 238000001514 detection method Methods 0.000 abstract description 13
- 230000012010 growth Effects 0.000 abstract description 7
- 239000000945 filler Substances 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 239000002207 metabolite Substances 0.000 abstract description 3
- 230000008635 plant growth Effects 0.000 abstract description 3
- 241001671621 Hemarthria altissima Species 0.000 abstract 1
- 238000010898 silica gel chromatography Methods 0.000 abstract 1
- 150000002500 ions Chemical class 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 239000000523 sample Substances 0.000 description 12
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 230000000694 effects Effects 0.000 description 8
- 238000010586 diagram Methods 0.000 description 7
- 244000233952 Polygonum bistorta Species 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- YWBHROUQJYHSOR-UHFFFAOYSA-N $l^{1}-selanylbenzene Chemical compound [Se]C1=CC=CC=C1 YWBHROUQJYHSOR-UHFFFAOYSA-N 0.000 description 3
- 235000007810 Eryngium campestre Nutrition 0.000 description 3
- 238000010268 HPLC based assay Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 235000018984 snakeroot Nutrition 0.000 description 3
- 235000007627 Caesalpinia Nutrition 0.000 description 2
- 241000522234 Caesalpinia Species 0.000 description 2
- 241001495493 Ophiorrhiza Species 0.000 description 2
- 241000925860 Senecio niveus Species 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 239000012159 carrier gas Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000012468 concentrated sample Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000001307 helium Substances 0.000 description 2
- 229910052734 helium Inorganic materials 0.000 description 2
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000000825 ultraviolet detection Methods 0.000 description 2
- FBDOJYYTMIHHDH-OZBJMMHXSA-N (19S)-19-ethyl-19-hydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]henicosa-2,4,6,8,10,14,20-heptaen-18-one Chemical compound CC[C@@]1(O)C(=O)OCC2=CN3Cc4cc5ccccc5nc4C3C=C12 FBDOJYYTMIHHDH-OZBJMMHXSA-N 0.000 description 1
- QPFMBZIOSGYJDE-ZDOIIHCHSA-N 1,1,2,2-tetrachloroethane Chemical group Cl[13CH](Cl)[13CH](Cl)Cl QPFMBZIOSGYJDE-ZDOIIHCHSA-N 0.000 description 1
- 235000001405 Artemisia annua Nutrition 0.000 description 1
- 240000000011 Artemisia annua Species 0.000 description 1
- 244000074881 Conyza canadensis Species 0.000 description 1
- 235000004385 Conyza canadensis Nutrition 0.000 description 1
- 235000015210 Fockea angustifolia Nutrition 0.000 description 1
- 244000186654 Fockea angustifolia Species 0.000 description 1
- 241000208152 Geranium Species 0.000 description 1
- OVSQVDMCBVZWGM-SJWGPRHPSA-N Hyperin Natural products O[C@H]1[C@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-SJWGPRHPSA-N 0.000 description 1
- FVQOMEDMFUMIMO-UHFFFAOYSA-N Hyperosid Natural products OC1C(O)C(O)C(CO)OC1OC1C(=O)C2=C(O)C=C(O)C=C2OC1C1=CC=C(O)C(O)=C1 FVQOMEDMFUMIMO-UHFFFAOYSA-N 0.000 description 1
- 241001477709 Ophiorrhiza japonica Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 235000014258 Polygonum bistorta Nutrition 0.000 description 1
- 235000009233 Stachytarpheta cayennensis Nutrition 0.000 description 1
- 101710183280 Topoisomerase Proteins 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- YBHILYKTIRIUTE-UHFFFAOYSA-N berberine Chemical compound C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 YBHILYKTIRIUTE-UHFFFAOYSA-N 0.000 description 1
- 229940093265 berberine Drugs 0.000 description 1
- QISXPYZVZJBNDM-UHFFFAOYSA-N berberine Natural products COc1ccc2C=C3N(Cc2c1OC)C=Cc4cc5OCOc5cc34 QISXPYZVZJBNDM-UHFFFAOYSA-N 0.000 description 1
- 239000002036 chloroform fraction Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000010201 enrichment analysis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229940089161 ginsenoside Drugs 0.000 description 1
- 229930182494 ginsenoside Natural products 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 238000009335 monocropping Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 229930000223 plant secondary metabolite Natural products 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- OVSQVDMCBVZWGM-DTGCRPNFSA-N quercetin 3-O-beta-D-galactopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-DTGCRPNFSA-N 0.000 description 1
- BBFYUPYFXSSMNV-UHFFFAOYSA-N quercetin-7-o-galactoside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=C2C(=O)C(O)=C(C=3C=C(O)C(O)=CC=3)OC2=C1 BBFYUPYFXSSMNV-UHFFFAOYSA-N 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000006257 total synthesis reaction Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B63/00—Purification; Separation; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C17/00—Preparation of halogenated hydrocarbons
- C07C17/38—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C17/00—Preparation of halogenated hydrocarbons
- C07C17/38—Separation; Purification; Stabilisation; Use of additives
- C07C17/389—Separation; Purification; Stabilisation; Use of additives by adsorption on solids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C209/00—Preparation of compounds containing amino groups bound to a carbon skeleton
- C07C209/82—Purification; Separation; Stabilisation; Use of additives
- C07C209/86—Separation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
- C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
- C07C29/76—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
- C07C37/004—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by obtaining phenols from plant material or from animal material
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/79—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/005—Processes comprising at least two steps in series
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/12—Purification; Separation; Use of additives by adsorption, i.e. purification or separation of hydrocarbons with the aid of solids, e.g. with ion-exchangers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
- C07D249/16—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms condensed with carbocyclic rings or ring systems
- C07D249/18—Benzotriazoles
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F11/00—Compounds containing elements of Groups 6 or 16 of the Periodic Table
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Water Supply & Treatment (AREA)
- Life Sciences & Earth Sciences (AREA)
- Botany (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a method for separating, enriching and detecting allelochemicals of a water culture root of a camptothecin-containing plant. Firstly, silica gel is used as a filling agent to fill a solid phase extraction column to extract and adsorb root system metabolites from a plant hydroponic liquid, then the filled silica gel is taken out and added to the upper layer of the silica gel chromatography column to carry out column chromatography separation, petroleum ether, ethyl acetate, ethanol and chloroform are used as eluents to carry out elution according to different combination ratios, eluent is collected according to eluent components, rotary evaporation concentration is carried out, and the separated components are analyzed by utilizing a gas chromatography-mass spectrometry (GC-MS) technology. The invention realizes the separation, enrichment and detection of allelochemicals in the water culture root exudates of two plants in large quantity. As a result, 25 allelochemicals were obtained by co-separation from the aqueous cultured root exudates of Camptotheca acuminata and Snake-grass. The invention has very important technical guidance function for detecting more allelochemicals in the hydroponics system, can remove substances with self-toxicity function in the allelochemicals in time based on the invention, ensures that the growth of plants cannot be inhibited, promotes the vigorous growth of the plants, and has important significance for optimizing the plant hydroponics system.
Description
Technical Field
The invention relates to the technical field of separation and preparation of compounds, in particular to a technology for separating and identifying a camptothecin-containing plant hydroponic root allelochemical substance.
Background
Camptothecin (CPT) is an important anticancer active alkaloid found in camptotheca acuminata, and was later found in a certain amount in plants of the genus ophiorrhiza. It is the only natural active ingredient found to date that exerts cytotoxicity by inhibiting topoisomerase. The market has great demand on camptothecin anticancer drugs, large-scale chemical total synthesis and in-vitro biosynthesis have great difficulty, and the large collection of plant resources such as camptotheca acuminata and the like to extract camptothecin is an important way for obtaining camptothecin at present. However, the long-term blind collection will cause the destruction of ecological environment and the waste of plant resources, which is not in accordance with the current environmental protection requirement. The biosynthesis means is currently used as a new way for continuously producing plant secondary metabolites. The hairy roots show the synthesis capability of secondary metabolites under the condition of in vitro culture, the yield of certain products is even higher than that of normal plants, and the hairy roots of medicinal plants such as camptotheca acuminata, sweet wormwood, south American wedeloa chinensis and the like are successfully cultured at home and abroad. Ginsenoside, berberine, etc. have been produced industrially by hairy root culture. Jiali ming and the like successfully collect root exudates in real time, continuously, enriched, quantified and accurately without damaging the root system under the cultivation condition by utilizing a circulating water root exudates collecting device, collect various amino acids in the root exudates within 7 days, and reach the detectable amount.
The applicant successfully establishes camptothecin-containing plant hydroponic systems such as camptotheca acuminata and Conyza canadensis and analyzes and confirms that root exudates of the hydroponic systems contain active metabolites such as camptothecin and hyperin. However, besides the active secondary metabolites with medicinal value, some allelochemicals with different activity functions, including allelochemicals with insecticidal activity, bacteriostatic activity, herbicidal activity, autotoxicity, etc., may also exist in the root exudates. The growth inhibition effect of the partial allelochemicals on plants brings certain negative effects on production, for example, the allelochemicals in root secretions cause continuous cropping obstacles of medicinal plants, phenolic acid compounds in the allelochemicals can influence metabolic pathways of plants such as rice and the like, and the allelochemicals generate autotoxicity phenomena so as to hinder growth. Therefore, the part of allelochemicals in the plant hydroponics root system secretion is accumulated for a long time to inhibit the growth of plants, more allelochemicals in the hydroponics system are proved, the substances with the self-toxicity function in the hydroponics system are removed in time, the growth of the plants is not inhibited, and the vigorous growth has important significance for optimizing the plant hydroponics system.
Disclosure of Invention
The invention aims to solve the technical problem of the technical defect of research on allelochemicals in the existing water culture system, and provides a method for separating and enriching the allelochemicals of the water culture root system of a camptothecin-containing plant.
The invention also aims to provide a method for detecting allelochemicals obtained by separation and enrichment.
The purpose of the invention is realized by the following technical scheme:
provides a method for separating and enriching allelochemicals of a water culture root system of a camptothecin-containing plant, which comprises the following steps:
s1, removing impurities from the collected hydroponic liquid, taking silica gel as a filling material, utilizing SPE solid phase extraction to enrich the hydroponic liquid until adsorption is balanced, collecting the silica gel containing root exudates after enrichment is finished, and drying to obtain silica gel powder containing a concentrated crude product;
s2, adding petroleum ether into the silica gel subjected to drying pretreatment, loading the silica gel into a column by a homogenization method, and washing the chromatographic column by using the petroleum ether;
s3, taking the silica gel powder containing the concentrated crude product obtained in the step S1, diluting and uniformly mixing the silica gel powder with petroleum ether, adding the silica gel powder into the chromatographic column prepared in the step S2, repeating the operation to ensure that the column body is fully compact, and then adding column chromatography silica gel on sample silica gel;
s4, preparing eluents with different proportions for gradient elution, and collecting effluent liquid in a segmented manner;
wherein, the camptothecin-containing plant is camptotheca acuminata or ophiorrhiza japonica; step S1, the hydroponic solution is the secretion of camptotheca acuminata hydroponic culture or the secretion of serpenthorum chinense hydroponic culture;
the eluent used for the secretion of the water culture of camptotheca acuminata is a mixture of petroleum ether, ethyl acetate and ethanol in the step S4; the eluent used for the secretion of the water culture of the serpentium chinense is a mixture of petroleum ether, ethanol, ethyl acetate and chloroform.
The invention designs a new separation and enrichment route, the root secretion water culture solution of the plant containing camptothecin is firstly used as a solid phase extraction filler to adsorb the root secretion, the adsorbent is taken out, and the water culture solution is directly added into a silica gel chromatographic column for elution without solvent elution and is subjected to rotary evaporation, so that the operation is simple, and the obtained separation and enrichment effect is better.
Preferably, the hydroponic solution in step S1 is the secreted solution of camptotheca acuminata after 60 days of hydroponic culture or the secreted solution of serpenthorum chinense after 45 days of hydroponic culture.
Preferably, step S1, the silica gel is replaced after the batch enrichment of the hydroponic fluid to the adsorption equilibrium. The drying is carried out at 45 ℃, and the mixture is stored for later use at 4 ℃.
Preferably, the particle size of the silica gel in the step S2 is 200-300 meshes. The pretreatment in step S2 is preferably carried out by baking at 110 ℃ for 2 hours.
Preferably, the above operation is repeated in step S3 to make the column fully compact, and then column chromatography silica gel with a thickness of 2cm is continuously added on the sample silica gel.
Preferably, the height of the homogenate-packed column in the step S4 is 30 cm; the step of flushing the chromatographic column with petroleum ether is to flush the chromatographic column with petroleum ether with the volume of 3-4 times of the column volume.
Preferably, the effluent fractional collection in step S4 is a total of 81 sample fractional collections, wherein effluent is collected every 50mL for samples No. 1-72, and effluent is collected every 100mL for samples No. 73-81.
Preferably, the eluent of the secretion of the camptotheca acuminate hydroponics in the step S4 is petroleum ether: ethyl acetate is a mixture of 1:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 by volume, or ethyl acetate: the volume ratio of the ethanol is 1:0, 9:1, 8:2, 7:3 and 6: 4.
Preferably, the allelochemicals separated and enriched from the exudate of the hydroponics of camptotheca acuminata are tropone, 4-oxo-7, 7-dinitro-4, 5,6, 7-tetrahydro (2H) benzotriazole, diphenylchromium, 1,2, 2-tetrachloroethane, 2, 4-di-tert-butylphenol, dibutyl phthalate, mono (2-ethylhexyl) phthalate, diisobutyl phthalate, N-methylaniline, aniline and N, N-dimethylaniline, respectively.
Preferably, the eluent of step S4, the eluent of the secretion of the hydroponic culture of the serpentis grass, is petroleum ether: a mixture of ethyl acetate in a volume ratio of 1:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1: 9; or ethanol: chloroform in a volume ratio of 0:1, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 1: 0.
Preferably, the allelochemicals separated and enriched from the exudate of the hydroponic culture of the serpenthorum is 1,1,2, 2-tetrachloroethane, N-dimethylaniline, diisobutyl phthalate, dibutyl phenylselenol phthalate, diphenylchromium, diisooctyl phthalate, di (2-ethylhexyl) phthalate, 2, 4-di-tert-butylphenol, 9-methylene-9H-fluorene, mono (2-ethylhexyl) phthalate, 3-methyl catechol, methyl palmitate, 2,2' -methylene bis- (4-methyl-6-tert-butylphenol) and dodecyl glycol respectively.
The invention also provides a detection method for separating the enriched allelochemicals, which comprises the steps of rotationally evaporating the effluent collected in the step S4 to dryness, adding acetonitrile, dissolving, filtering by an organic phase filter membrane, and then carrying out chromatographic analysis; preferably the rotary evaporation to dryness is rotary evaporation to dryness at 45 ℃; the organic phase filter membrane filtration is to use a 0.22 mu m organic phase filter membrane for filtration; the chromatographic analysis is analysis identification by GC-MS.
On this basis, most preferably, the separation and enrichment method comprises the following steps:
s1, removing impurities from the collected hydroponic liquid, taking silica gel as a filling material, utilizing SPE solid phase extraction to enrich the hydroponic liquid until adsorption is balanced, collecting the silica gel containing root exudates after enrichment is finished, and drying to obtain silica gel powder containing a concentrated crude product;
s2, adding petroleum ether into the pretreated silica gel, loading the silica gel into a column by a homogenization method, and washing the chromatographic column by the petroleum ether. The pretreatment in step S2 is preferably carried out by baking at 110 ℃ for 2 hours.
S3, taking the silica gel powder containing the concentrated crude product obtained in the step S1, diluting and uniformly mixing the silica gel powder with petroleum ether, adding the silica gel powder into the chromatographic column prepared in the step S2, repeating the operation to ensure that the column body is fully compact, and then adding column chromatography silica gel on sample silica gel;
s4, preparing eluents with different proportions for gradient elution, and collecting effluent liquid in a segmented manner;
s5, the effluent collected in the step S4 is rotated to be evaporated to dryness, acetonitrile is added for dissolution, and chromatographic analysis is carried out after the filtration of an organic phase filter membrane.
The invention has the beneficial effects that:
the invention provides a novel method for separating and enriching allelochemicals from water culture root exudates of plants containing camptothecin, which obtains 25 allelochemicals in total by further aiming and precise optimization of separation and enrichment means and conditions, and the number of the allelochemicals separated and enriched from any one plant of camptotheca acuminata or ophiorrhiza serrulata exceeds more than 10. The invention has very important technical guidance function for detecting allelochemicals in the hydroponics system, can remove substances with self-toxicity function in the allelochemicals in time based on the invention, ensures that the growth of plants cannot be inhibited, promotes the vigorous growth of the plants, and has important significance for optimizing the plant hydroponics system.
The method has simple and easy steps, firstly, silica gel is used as a filling agent to fill a solid phase extraction column to extract and adsorb the root system metabolites from the plant hydroponic liquid, then the filled silica gel is taken out and added to the upper layer of the silica gel chromatographic column to carry out column chromatography separation, then different combinations of petroleum ether, ethyl acetate, ethanol and chloroform are used as eluents to elute in different proportions of different combinations, the eluents are collected according to the components of the eluents and are evaporated and concentrated in a rotary mode, and the separation components are analyzed by further scientifically utilizing a gas chromatography-mass spectrometry (GC-MS) technology aiming at the separation method, so that the large-scale separation, enrichment and detection of allelochemicals in the two plant hydroponic root system secretions are realized.
The invention detects more allelochemicals from two camptothecin-containing plant hydroponic root secretions, including 11 allelochemicals such as 1,1,2, 2-tetrachloroethane, N-dimethylaniline, diphenylchromium, 2, 4-di-tert-butylphenol, dibutyl phthalate, mono (2-ethylhexyl) phthalate, diisobutyl phthalate and the like detected from the camptothecin hydroponic root secretions; 14 allelochemicals such as dibutyl phthalate, mono (2-ethylhexyl) phthalate, diisobutyl phthalate, phenylselenol, diphenylchromium and the like are detected in the root secretion of the Guangzhou snakeweed hydroponics.
Drawings
FIG. 1 shows petroleum ether of hydroponic root system of Geranium serpentium: and (3) detecting ethyl acetate by HPLC (high performance liquid chromatography).
FIG. 2 ethanol of hydroponic root system of S.ophioides: HPLC assay of chloroform 2:8 fractions.
FIG. 3 ethanol of hydroponic root system of S.ophioides: HPLC assay of 4:6 chloroform fraction.
FIG. 4 ethanol of hydroponic root system of S.ophioides: HPLC assay of chloroform 6:4 fraction.
FIG. 5 shows HPLC detection results of ethanol component of hydroponic roots of S.niveus.
FIG. 6 shows petroleum ether of hydroponic roots of S.ophioides: and (3) performing a GC-MS (gas chromatography-mass spectrometry) total ion flow diagram on the ethyl acetate-7: 3 component.
FIG. 7 ethanol of hydroponic root system of S.ophioides: a 2:8 component GC-MS total ion flow diagram.
FIG. 8 ethanol of hydroponic root system of S.ophioides: a GC-MS total ion flow diagram of chloroform-4: 6 components.
FIG. 9 ethanol of hydroponic root system of S.ophioides: GC-MS total ion flow diagram of chloroform 6:4 components.
FIG. 10 GC-MS total ion flow graph of ethanol component of hydroponic rootstock of S.niveus.
FIG. 11 petroleum ether, a root secretion of Camptotheca acuminata: HPLC detection of ethyl acetate (9:1) fractions.
FIG. 12 petroleum ether, a root secretion of Camptotheca acuminata: GC-MS total ion flow diagram of the ethyl acetate (9:1) component.
Detailed Description
The process of the invention is further illustrated below with reference to specific examples and figures. The following examples are for illustrative purposes only and are not to be construed as limiting the invention. Unless otherwise specified, the biomaterials, reagent raw materials used in the following examples are conventionally commercially available or commercially available biomaterials and reagent raw materials, and unless otherwise specified, the methods and apparatuses used in the following examples are those conventionally used in the art. Example 1 enrichment and isolation of allelochemicals from hydroponic roots of Serpentis grass
1. Construction of snakeroot grass hydroponic system
1.1 hydroponic culture of Serpentis grass and related operations
After the Guangzhou snakeroot is collected, selecting a plant with good growth vigor, slightly washing the plant with tap water to remove soil at the root, avoiding damaging the root as much as possible, putting the plant into a 6L plastic pot, adding water into the plastic pot, culturing in an artificial greenhouse, wherein 6-8 plants are planted in each pot, and the Guangzhou snakeroot is suitable for growing under a continuously ventilated shading greenhouse at the temperature of 18-25 ℃.
1.2 enrichment of root exudate
And after water culture is carried out for 45 days, combining and collecting water culture solutions, filtering to remove solid impurities, taking silica gel as a filler, carrying out solid-phase extraction by using SPE (solid phase extraction), enriching the water culture solutions in batches until adsorption balance is reached, replacing the silica gel, collecting the silica gel containing root exudates after the enrichment is finished, drying at 45 ℃, and storing at 4 ℃ for later use.
1.3 separation and detection process of concentrated solution
(1) Column loading and sample loading
Adding petroleum ether into 200-300-mesh pretreated (dried for 2 hours at 110 ℃), loading the silica gel into a column by a homogenization method, washing the column by using petroleum ether with the height being about 30cm and the volume being 3-4 times of the column volume, diluting and uniformly mixing 3-4 g of silica gel powder containing a concentrated crude product with a small amount of petroleum ether, slowly adding the silica gel powder into a chromatographic column, and repeating the operation to fully compact the column. And continuously adding 2cm of column chromatography silica gel on the silica gel sample (preventing the silica gel sample from being stirred to cause plane damage when liquid is added).
(2) Gradient elution
Different proportions of eluents (shown in Table 1) were prepared and gradient elution was carried out, the amount and polarity of the eluents being determined by the HPLC analysis results. The effluent is collected in stages and recorded, and the effluent is collected once every 40mL, the collected effluent is transferred to a pear-shaped bottle, the solution is evaporated to dryness by rotating at 35 ℃, 1.5mL of acetonitrile is added, and the solution is dissolved by ultrasonic oscillation. The resulting mixture was filtered through a 0.22 μm organic phase filter and subjected to chromatography.
TABLE 1 eluent ratio and dosage
2. Analysis of the eluate:
2.1HPLC detection
Chromatographic conditions are as follows: the chromatographic column is Kromasil100-5C18(4.6mm × 250mm, 5 μm); ultraviolet detection wavelength 254 nm; the mobile phase was acetonitrile/water (V/V, 40/60)), which was degassed ultrasonically before use and filtered through a 0.22 μm filter at a flow rate of 1 mL/min; the column temperature is 28 ℃; the amount of the sample was 20. mu.L.
The invention carries out gradient elution by different proportions of ethyl acetate and petroleum ether, ethanol and chloroform, the eluent is firstly detected by HPLC, and the ratio of petroleum ether: ethyl acetate (7:3), ethanol: chloroform (2:8), ethanol: chloroform (4:6), ethanol: chloroform (6:4), ethanol: chloroform (1:0), and the chemoattractant has a relatively good separation effect, as shown in fig. 1,2, 3, 4, and 5.
2.2GC-MS detection
Chromatographic conditions are as follows: chromatography column 30m × 0.25mm, 0.33 μmSE-30 elastic quartz capillary column. Temperature programming: the initial temperature is 50 deg.C, maintained for 2min, increased to 150 deg.C at a rate of 5 deg.C/min, maintained for 5min, increased to 220 deg.C at a rate of 5 deg.C/min, and maintained for 10 min. The injection port temperature is 250 ℃, the carrier gas is helium, the split ratio is 40:1, the column front pressure is 68kpa, and the injection amount is 1 muL.
Mass spectrum conditions: the ion source EI has the ion source temperature of 230 ℃, the electron energy of 70eV, the interface temperature of 270 ℃, the solvent delay of 3min and the ion scanning range of 50-800 amu.
The eluent concentrated sample with better separation degree and single peak shape after HPLC preliminary analysis is screened, namely petroleum ether: ethyl acetate (7:3), ethanol: chloroform (2:8), ethanol: chloroform (4:6), ethanol: chloroform (1: 0). Detecting a heat-stable compound in the plant root secretion by GC-MS, and exploring allelochemicals in the plant root secretion. The ion flow diagrams are shown in fig. 6,7, 8, 9 and 10. The unit of time in fig. 6 is min. The units of time are in min unless otherwise specified in the figures.
3. And (3) analysis results:
the Caesalpinia serrulata root secretion exudate is analyzed by GC-MS, and 14 allelochemicals are separated and detected in the Caesalpinia serrulata root secretion and are respectively 1,1,2, 2-tetrachloroethane, N-dimethylaniline, diisobutyl phthalate, dibutyl phthalate phenylselenol, diphenylchromium, diisooctyl phthalate, di (2-ethylhexyl) phthalate, 2, 4-di-tert-butylphenol, 9-methylene-9H-fluorene, mono (2-ethylhexyl) phthalate, methyl palmitate, 3-methyl catechol, 2,2' -methylene bis- (4-methyl-6-tert-butylphenol) and dodecyl glycol.
Wherein the weight ratio of petroleum ether: ethyl acetate, ethanol: eluting with chloroform at different ratio, and separating to obtain maximum allelochemicals (8 kinds) when the eluent is ethanol and chloroform at a ratio of 1:0, and separating to obtain 5 kinds of allelochemicals when the eluent is ethanol and chloroform at a ratio of 2:8 and ethanol and chloroform at a ratio of 4: 6.
Example 2 enrichment analysis of root exudate allelochemicals of Camptotheca acuminata
1. Construction of camptotheca water culture system
1.1 Camptotheca acuminata hydroponic culture related operations
The exploration of the construction of the water culture system of the camptotheca acuminate seedling: selecting about 50cm of seedling with excellent growth condition, and digging without damaging roots. And cleaning soil at roots, and culturing outdoors.
1.2 enrichment of root exudate
After 60 days of water culture, the water culture solution is collected and filtered to remove solid impurities. Silica gel is used as a filling material, SPE solid phase extraction is utilized, the water culture solution is enriched in batches until adsorption balance is achieved, and the silica gel is replaced. Collecting the silica gel containing root exudates after the enrichment is finished, drying at 45 ℃, and storing at 4 ℃ for later use.
1.3 separation and detection process of concentrated solution
(1) Column loading and sample loading
Taking 200-300 meshes of pretreated silica gel (dried for 2 hours at 110 ℃), adding petroleum ether, loading the silica gel into a column by a homogenization method, washing the column by using petroleum ether with the column volume being 3-4 times of that of the column, taking 20mL of silica gel powder of a concentrated crude product, diluting and uniformly mixing the silica gel powder with a small amount of petroleum ether, slowly adding the silica gel powder into a chromatographic column, repeating the operation to ensure that the column is fully compact, and continuously adding column chromatography silica gel with the thickness of 2cm onto the sample silica gel (preventing the plane from being damaged due to the excitation of the silica gel sample during liquid adding).
(2) Gradient elution
Different proportions of eluents (table 2) were prepared and gradient elution was carried out, each time 50mL of eluent was added, and for effective separation and purification, the increasing degree and amount of the solvent polarity were mainly determined by HPLC chromatography detection of each fraction of the effluent. And (4) collecting the effluent liquid in a segmented mode and recording, wherein the effluent liquid is collected once every 50mL for No. 1-72 samples and once every 100mL for No. 73-81 samples, transferring the collected effluent liquid to a pear-shaped bottle, and rotating at 45 ℃ to evaporate to dryness. 1.5mL of acetonitrile was added and dissolved by ultrasonic oscillation. The resulting mixture was filtered through a 0.22 μm organic phase filter and subjected to chromatography.
TABLE 2 eluent ratio and dosage
2. Analysis of the eluate:
2.1HPLC detection
Chromatographic conditions are as follows: the chromatographic column is Kromasil100-5C18(4.6mm × 250mm, 5 μm); ultraviolet detection wavelength 254 nm; the mobile phase is acetonitrile/water (V/V, 40/60), and is degassed by ultrasound before use, and filtered through 0.22 μm filter membrane at flow rate of 1 mL/min; the column temperature is 28 ℃; the amount of the sample was 20. mu.L.
The invention carries out gradient elution by different proportions of ethyl acetate and petroleum ether and ethyl acetate and ethanol, the eluent is firstly detected by HPLC, and after detection, the ratio of the ethyl acetate to the petroleum ether: ethyl acetate (1:0), petroleum ether: ethyl acetate (9:1), petroleum ether: ethyl acetate (7:3), petroleum ether: ethyl acetate (3:7), ethyl acetate: ethanol (8:2), ethyl acetate: ethanol (6:4), the allelochemicals separation effect is better. Such as in the case of petroleum ether: the liquid phase analysis of ethyl acetate (9:1) showed a clear separation effect, as shown in FIG. 11.
2.2GC-MS detection
Chromatographic conditions are as follows: chromatographic column 30m × 0.25mm, 0.33 μmSE-30 elastic quartz capillary column temperature program: the initial temperature is 50 deg.C, maintained for 2min, increased to 150 deg.C at a rate of 5 deg.C/min, maintained for 5min, increased to 220 deg.C at a rate of 5 deg.C/min, and maintained for 10 min. The injection port temperature is 250 ℃, the carrier gas is helium, the split ratio is 40:1, the column front pressure is 68kpa, and the injection amount is 1 muL.
Mass spectrum conditions: the ion source EI has the ion source temperature of 230 ℃, the electron energy of 70eV, the interface temperature of 270 ℃, the solvent delay of 3min and the ion scanning range of 50-800 amu.
The eluent concentrated sample with better separation degree and single peak shape after HPLC preliminary analysis is screened, namely petroleum ether: ethyl acetate (1:0), petroleum ether: ethyl acetate (9:1), petroleum ether: ethyl acetate (7:3), petroleum ether: ethyl acetate (3:7), ethyl acetate: ethanol (8:2), ethyl acetate: ethanol (6:4), the allelochemicals separation effect is better. These several elution ratios. Detecting a heat-stable compound in the plant root secretion by GC-MS, and exploring allelochemicals in the plant root secretion. Wherein when the eluent is petroleum ether: when ethyl acetate is 9:1, most allelochemicals are separated, and the ion flow diagram is shown in fig. 12, wherein the number of allelochemicals is 4.
3. And (3) analysis results:
by analyzing the water culture root secretion of the camptotheca acuminate by GC-MS, 11 allelochemicals are detected, namely tropone, 4-oxo-7, 7-dinitro-4, 5,6, 7-tetrahydro (2H) benzotriazole, diphenylchromium, 1,2, 2-tetrachloroethane, 2, 4-di-tert-butylphenol, dibutyl phthalate, mono (2-ethylhexyl) phthalate, diisobutyl phthalate, N-methylaniline, aniline and N, N-dimethylaniline.
Claims (7)
1. A method for separating and enriching allelochemicals of a water culture root of a camptothecin-containing plant is characterized by comprising the following steps:
s1, removing impurities from the collected hydroponic liquid, taking silica gel as a filling material, utilizing SPE solid phase extraction to enrich the hydroponic liquid until adsorption is balanced, collecting the silica gel containing root exudates after enrichment is finished, and drying to obtain silica gel powder containing a concentrated crude product;
s2, adding petroleum ether into the silica gel subjected to drying pretreatment, loading the silica gel into a column by a homogenization method, and washing the chromatographic column by using the petroleum ether;
s3, taking the silica gel powder containing the concentrated crude product obtained in the step S1, diluting and uniformly mixing the silica gel powder with petroleum ether, adding the silica gel powder into the chromatographic column prepared in the step S2, repeating the operation to ensure that the column body is fully compact, and then adding column chromatography silica gel on sample silica gel;
s4, preparing eluents with different proportions for gradient elution, and collecting effluent liquid in a segmented manner;
wherein, the water culture solution of the step S1 is the secretion of the water culture of camptotheca acuminate or the secretion of the water culture of serpentphone grass;
the eluent used in the secretion of the water culture of camptotheca acuminata in the step S4 is a mixture of petroleum ether, ethyl acetate and ethanol, wherein the ratio of petroleum ether: ethyl acetate in a volume ratio of 1:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, ethyl acetate: the volume ratio of the ethanol is 1:0, 9:1, 8:2, 7:3 and 6: 4; eluent used for the secretion of the water culture of the serpentium chinense is a mixture of petroleum ether, ethanol, ethyl acetate and chloroform, wherein the ratio of petroleum ether: ethyl acetate in a volume ratio of 1:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, ethanol: the volume ratio of chloroform is 0:1, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4 and 7; 3. 8:2, 9:1, 1: 0; separating and enriching allelochemicals from the secretion of water culture of Camptotheca acuminata respectively including tropone, 4-oxo-7, 7-dinitro-4, 5,6, 7-tetrahydro-2H-benzotriazole, chromium diphenyl, 1,2, 2-tetrachloroethane, 2, 4-di-tert-butylphenol, dibutyl phthalate, mono (2-ethylhexyl) phthalate, diisobutyl phthalate, N-methylaniline, aniline and N, N-dimethylaniline; the allelochemicals separated and enriched from the secretion of the serpenthorum hydroponics are 1,1,2, 2-tetrachloroethane, N-dimethylaniline, diisobutyl phthalate, dibutyl phthalate selenophenol, diphenyl chromium, diisooctyl phthalate, di (2-ethylhexyl) phthalate, 2, 4-di-tert-butylphenol, 9-methylene-9H-fluorene, mono (2-ethylhexyl) phthalate, 3-methyl catechol, methyl palmitate, 2,2' -methylene bis- (4-methyl-6-tert-butylphenol) and dodecyl glycol respectively.
2. The method for separating and enriching allelochemicals from the water culture root of camptothecin-containing plants as claimed in claim 1, wherein the water culture solution of step S1 is the secretion solution of camptotheca acuminata after 60 days water culture or the secretion solution of serpenthorum chinense after 45 days water culture.
3. The method for separating and enriching the hydroponic root chemoattractant substances of the camptothecin-containing plants as claimed in claim 1, wherein the silica gel in step S2 has a particle size of 200-300 meshes.
4. The method for separating and enriching the hydroponic root chemoattractant material of the camptothecin-containing plant as claimed in claim 1, wherein the height of the column packed by the homogenate method in step S4 is 30 cm; the step of flushing the chromatographic column with petroleum ether is to flush the chromatographic column with petroleum ether with the volume of 3-4 times of the column volume.
5. The method for separating and enriching the hydroponic allelochemicals of the camptothecin-containing plants as claimed in claim 1, wherein the step of step S4 comprises collecting 81 samples, wherein the effluent is collected every 50mL for samples No. 1-72, and the effluent is collected every 100mL for samples No. 73-81.
6. The method for separating and enriching the chemoattractant materials of the hydroponic root of the camptothecin-containing plants as claimed in any one of claims 1 to 5, further comprising the steps of evaporating the effluent collected in step S4 to dryness by rotation, adding acetonitrile, dissolving, filtering with an organic phase filter, and then performing chromatography.
7. The method for separating and enriching the camptothecin-containing plant hydroponic allelochemicals as claimed in claim 6, wherein the rotary evaporation to dryness is rotary evaporation to dryness at 45 ℃; the organic phase filter membrane filtration is to use a 0.22 mu m organic phase filter membrane for filtration;
the chromatographic analysis is analysis identification by GC-MS.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710425639.8A CN107417479B (en) | 2017-06-07 | 2017-06-07 | Method for separating and enriching allelochemicals of water culture root of camptothecin-containing plants |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710425639.8A CN107417479B (en) | 2017-06-07 | 2017-06-07 | Method for separating and enriching allelochemicals of water culture root of camptothecin-containing plants |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107417479A CN107417479A (en) | 2017-12-01 |
| CN107417479B true CN107417479B (en) | 2020-11-06 |
Family
ID=60428725
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710425639.8A Active CN107417479B (en) | 2017-06-07 | 2017-06-07 | Method for separating and enriching allelochemicals of water culture root of camptothecin-containing plants |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107417479B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101376659A (en) * | 2008-09-27 | 2009-03-04 | 上海北卡医药技术有限公司 | Method for purifying camptothecine and derivative thereof |
-
2017
- 2017-06-07 CN CN201710425639.8A patent/CN107417479B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101376659A (en) * | 2008-09-27 | 2009-03-04 | 上海北卡医药技术有限公司 | Method for purifying camptothecine and derivative thereof |
Non-Patent Citations (2)
| Title |
|---|
| 喜树水培体系中喜树碱、及其他组分的初步鉴定;程杏安等;《中国农学通报》;20141231;第30卷(第1期);第37-41段 * |
| 蛇根草水培体系中喜树碱、金丝桃苷及其他组分的初步鉴定;程杏安;《广东农业科学》;20131231(第19期);第94-98页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107417479A (en) | 2017-12-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2002534422A (en) | Method for high yield extraction of paclitaxel from paclitaxel-containing materials | |
| CN102924467B (en) | Preparation method for extracting and purifying bruceine D form brucea javanica | |
| CN102321135A (en) | Method for separating and purifying cordycepin by utilizing high-speed counter-current chromatography | |
| CN105585578B (en) | A kind of preparation method of rapamycin | |
| CN107417479B (en) | Method for separating and enriching allelochemicals of water culture root of camptothecin-containing plants | |
| CN104829474A (en) | Method for preparing glycine betaine chemical reference substances from boxthorn leaves | |
| CN105175426B (en) | A kind of method of the extraction purification Bergenin from treebine stem | |
| CN112321664B (en) | Method for extracting, separating and purifying inonotus obliquus alcohol | |
| CN101278989A (en) | Method for extracting Thalictrum aquilegifolium total alkaloids and total saponins from Thalictrum aquilegifolium | |
| CN109609568B (en) | Method for preparing swainsonine by solid fermentation | |
| CN111234559B (en) | Extraction and purification method of purple locust pigment | |
| CN109810014A (en) | A kind of two caffeoyl spermidine class compound selective enrichment methods in fructus lycii | |
| CN107141198A (en) | A kind of method that phytol is extracted in the ballstone algae from ocean | |
| CN112778095A (en) | Extraction process of resveratrol in roselle leaves | |
| JP3336024B2 (en) | Method for producing plant metabolites | |
| CN110623995A (en) | Method for extracting total flavonoids from aerial parts of liquorice and preparing liposome of total flavonoids | |
| CN104672090A (en) | Method for separating and purifying ramie leaf chlorogenic acid | |
| CN106866318A (en) | The growth substrate of arithoke cellulose content in a kind of raising leaf of Cynara scolymus L | |
| CN105228638A (en) | Be rich in the extract of the Papilio xuthus (Linne)． Hipeastrum vittalum (L Her.) Herb.-Amaryllisvittata Ait. of galantamine | |
| CN114933632B (en) | Method for preparing 5 kinds of ustilaginoidea virens toxins through simultaneous separation and purification | |
| CN102408455A (en) | Plant-derived zinc-fixation compound and preparation method and application thereof | |
| CN116102571B (en) | Separation and purification method of ocean fat-soluble toxin component in protonema reticulata | |
| CN101928291A (en) | Method for separating and purifying ansamitocin from precious orange synnema actinomycetes fermentation liquor | |
| CN109867650B (en) | Preparation method for extracting allelochemical Salcolin A from highland barley straws | |
| CN107857762B (en) | Method for extracting and preparing 6-benzylaminopurine in concentrated biogas slurry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Liu Zhanmei Inventor after: Cheng Xingan Inventor after: Jiang Xuhong Inventor after: Lin Xianwei Inventor before: Cheng Xingan Inventor before: Liu Zhanmei Inventor before: Jiang Xuhong Inventor before: Lin Xianwei |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |