CN106046081B - The preparation and application of flavonoid glycoside active component in a kind of polygonum capitatum - Google Patents
The preparation and application of flavonoid glycoside active component in a kind of polygonum capitatum Download PDFInfo
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- CN106046081B CN106046081B CN201610408978.0A CN201610408978A CN106046081B CN 106046081 B CN106046081 B CN 106046081B CN 201610408978 A CN201610408978 A CN 201610408978A CN 106046081 B CN106046081 B CN 106046081B
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- flavonoid glycoside
- methanol
- polygonum capitatum
- phytophthora
- active component
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- 229930182486 flavonoid glycoside Natural products 0.000 title claims abstract description 45
- 150000007955 flavonoid glycosides Chemical class 0.000 title claims abstract description 30
- 241000764065 Persicaria capitata Species 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- -1 flavonoid glycoside compounds Chemical class 0.000 claims abstract description 19
- 238000000926 separation method Methods 0.000 claims abstract description 15
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000000575 pesticide Substances 0.000 claims abstract description 7
- 239000003814 drug Substances 0.000 claims abstract description 5
- 238000000746 purification Methods 0.000 claims abstract description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000741 silica gel Substances 0.000 claims abstract description 4
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 87
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 48
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 26
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 claims description 22
- 150000001875 compounds Chemical class 0.000 claims description 15
- 239000002904 solvent Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims description 12
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 11
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 claims description 11
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 claims description 11
- 235000005875 quercetin Nutrition 0.000 claims description 11
- 229960001285 quercetin Drugs 0.000 claims description 11
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 10
- 150000008265 rhamnosides Chemical class 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 9
- SOSLMHZOJATCCP-AEIZVZFYSA-N afzelin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC1=C(C=2C=CC(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O SOSLMHZOJATCCP-AEIZVZFYSA-N 0.000 claims description 8
- SOSLMHZOJATCCP-PADPQNGGSA-N afzelin Natural products O([C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](C)O1)C1=C(c2ccc(O)cc2)Oc2c(c(O)cc(O)c2)C1=O SOSLMHZOJATCCP-PADPQNGGSA-N 0.000 claims description 8
- 239000000284 extract Substances 0.000 claims description 8
- HEAQNIWCWRHODF-UHFFFAOYSA-N kaempferol 3-O-alpha-L-rhamnopyranoside Natural products OC1C(O)C(O)C(C)OC1OC1=C(C=2C=CC=CC=2)OC2=CC(O)=CC(O)=C2C1=O HEAQNIWCWRHODF-UHFFFAOYSA-N 0.000 claims description 8
- LPEPTRFUOKMJCH-UHFFFAOYSA-N myricetin 7-O-alpha-L-rhamnopyranoside Natural products OC1C(O)C(O)C(C)OC1OC1=CC(O)=C2C(=O)C(O)=C(C=3C=C(O)C(O)=C(O)C=3)OC2=C1 LPEPTRFUOKMJCH-UHFFFAOYSA-N 0.000 claims description 8
- OVSQVDMCBVZWGM-IDRAQACASA-N Hirsutrin Natural products O([C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1)C1=C(c2cc(O)c(O)cc2)Oc2c(c(O)cc(O)c2)C1=O OVSQVDMCBVZWGM-IDRAQACASA-N 0.000 claims description 7
- 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 claims description 7
- GXMWXESSGGEWEM-UHFFFAOYSA-N isoquercitrin Natural products OCC(O)C1OC(OC2C(Oc3cc(O)cc(O)c3C2=O)c4ccc(O)c(O)c4)C(O)C1O GXMWXESSGGEWEM-UHFFFAOYSA-N 0.000 claims description 7
- MSBHOJSEFZPTET-UHFFFAOYSA-N isorhamnetin 3-O-galactoside Natural products OCC1OC(OC2=C(Oc3c(O)ccc(O)c3C2=O)c4ccc(O)c(O)c4)C(O)C(O)C1O MSBHOJSEFZPTET-UHFFFAOYSA-N 0.000 claims description 7
- OVSQVDMCBVZWGM-QSOFNFLRSA-N quercetin 3-O-beta-D-glucopyranoside 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-QSOFNFLRSA-N 0.000 claims description 7
- KRXANNXAPJODKJ-UHFFFAOYSA-N quercetin 3-O-beta-D-glucopyranoside Natural products OCC1OC(COC2=C(Oc3cc(O)cc(O)c3C2=O)c4ccc(O)c(O)c4)C(O)C(O)C1O KRXANNXAPJODKJ-UHFFFAOYSA-N 0.000 claims description 7
- 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 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 239000003125 aqueous solvent Substances 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 3
- 238000003810 ethyl acetate extraction Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000004007 reversed phase HPLC Methods 0.000 claims description 3
- 238000003809 water extraction Methods 0.000 claims description 3
- 210000000692 cap cell Anatomy 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000003480 eluent Substances 0.000 claims description 2
- 238000002386 leaching Methods 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims 1
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 claims 1
- 241000233614 Phytophthora Species 0.000 abstract description 21
- 239000002775 capsule Substances 0.000 abstract description 15
- 230000002401 inhibitory effect Effects 0.000 abstract description 9
- 238000000605 extraction Methods 0.000 abstract description 3
- 238000012827 research and development Methods 0.000 abstract description 2
- 230000000975 bioactive effect Effects 0.000 abstract 1
- 238000003808 methanol extraction Methods 0.000 abstract 1
- 230000002265 prevention Effects 0.000 abstract 1
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 239000000203 mixture Substances 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 238000005160 1H NMR spectroscopy Methods 0.000 description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 5
- 238000001425 electrospray ionisation time-of-flight mass spectrometry Methods 0.000 description 5
- 229910052739 hydrogen Inorganic materials 0.000 description 5
- 239000001257 hydrogen Substances 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 239000005807 Metalaxyl Substances 0.000 description 3
- 241000233616 Phytophthora capsici Species 0.000 description 3
- 241001377695 Polygonum arenastrum Species 0.000 description 3
- 235000006386 Polygonum aviculare Nutrition 0.000 description 3
- 230000011681 asexual reproduction Effects 0.000 description 3
- 238000013465 asexual reproduction Methods 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- ZQEIXNIJLIKNTD-UHFFFAOYSA-N methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)alaninate Chemical compound COCC(=O)N(C(C)C(=O)OC)C1=C(C)C=CC=C1C ZQEIXNIJLIKNTD-UHFFFAOYSA-N 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 206010059866 Drug resistance Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 206010035148 Plague Diseases 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- IYRMWMYZSQPJKC-UHFFFAOYSA-N kaempferol Chemical compound C1=CC(O)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 IYRMWMYZSQPJKC-UHFFFAOYSA-N 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- 230000002087 whitening effect Effects 0.000 description 2
- 150000008505 β-D-glucopyranosides Chemical class 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- UBSCDKPKWHYZNX-UHFFFAOYSA-N Demethoxycapillarisin Natural products C1=CC(O)=CC=C1OC1=CC(=O)C2=C(O)C=C(O)C=C2O1 UBSCDKPKWHYZNX-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- IKMDFBPHZNJCSN-UHFFFAOYSA-N Myricetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC(O)=C(O)C(O)=C1 IKMDFBPHZNJCSN-UHFFFAOYSA-N 0.000 description 1
- 241000219050 Polygonaceae Species 0.000 description 1
- CDMXXXZRZWQJQE-UHFFFAOYSA-N acetic acid;2-methylprop-2-enoic acid Chemical compound CC(O)=O.CC(=C)C(O)=O CDMXXXZRZWQJQE-UHFFFAOYSA-N 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 239000012675 alcoholic extract Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000012272 crop production Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 239000000417 fungicide Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 235000008777 kaempferol Nutrition 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000000401 methanolic extract Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- UXOUKMQIEVGVLY-UHFFFAOYSA-N morin Natural products OC1=CC(O)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UXOUKMQIEVGVLY-UHFFFAOYSA-N 0.000 description 1
- 208000030194 mouth disease Diseases 0.000 description 1
- PCOBUQBNVYZTBU-UHFFFAOYSA-N myricetin Natural products OC1=C(O)C(O)=CC(C=2OC3=CC(O)=C(O)C(O)=C3C(=O)C=2)=C1 PCOBUQBNVYZTBU-UHFFFAOYSA-N 0.000 description 1
- 235000007743 myricetin Nutrition 0.000 description 1
- 229940116852 myricetin Drugs 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Saccharide Compounds (AREA)
Abstract
The present invention provides a kind of preparation method of polygonum capitatum flavonoid glycoside active component, by the way that polygonum capitatum is crushed, methanol extraction, filter concentration, it is first open post separation through silica gel chromatograph, then is obtained by the octadecyl silane column purification that is open, this component main component is five flavonoid glycoside compounds.The present invention establishes the bioactive systems for inhibiting phytophthora asexual spore capsule to generate, and by the system, finds this polygonum capitatum flavonoid glycoside component and wherein five flavonoid glycoside compounds can significantly inhibit the generation of phytophthora asexual spore capsule.Polygonum capitatum flavonoid glycoside active component provided by the invention can be applied in the pesticide of preparation prevention and treatment plant phytophthora root rot.Preparation method of the present invention is simple, and the product purity of extraction is high, and polygonum capitatum is the cheap Chinese medicine that is easy to get, and increases the medicinal usage of polygonum capitatum, and the research and development for anti-phytophthora pesticide are laid a good foundation.
Description
Technical field
The invention belongs to pharmaceutical technology field, it is related to spending flavonoid glycoside in flavonoid glycoside active component in knotweed, more particularly to flower knotweed living
The preparation method of property component and preparing the application in resisting phytophthora disease pesticide.
Background technique
According to the United Nations's data statistics, world population is just increasing year by year, it is contemplated that will rise to 8.9 to the year two thousand fifty world population
Hundred million, and effectively cultivated area is constantly being reduced, it is therefore desirable to more foods are produced on less cultivated area to avoid
It is a wide range of hungry.In the numerous threats for influencing crop production, the occurrence frequency of plant disease is high, caused by lose it is also the tightest
Weight.Wherein phytophthora can cause most destructive plant disease in the world, not only endanger agricultural production, but also destroy nature
The ecosystem.In addition, chemical pesticide is long-term and is widely applied so that most of phytophthoras produce seriously many fungicide
Drug resistance.Therefore, find and find that new anti-phytophthora active material has become urgent problem.
Mycelia growth and zoospore is inhibited to sprout two sides currently, anti-phytophthora active matter Quality Research is concentrated mainly on
Face inhibits the active matter Quality Research of phytophthora asexual reproduction phases sporangium generation few to having.And the generation of sporangium
It is the critical period of phytophthora asexual reproduction phases, the zoospore generated is the basis of phytophthora sprawling, therefore finds tool
There is the natural active matter for inhibiting asexual spore capsule to generate, may find that new action target spot, help to alleviate or solves to work as
Preceding increasingly serious drug resistance pushes the development and utilization of vegetable plague protective agents.
Polygonum capitatum (Polygonum capitatum) belongs to polygonaceae herbaceos perennial, is the famous medicinal plant in China
Object.Its medical value also existing research: Herba Polygoni Capitati extract and its application in hypoglycemic drug；Herba Polygoni Capitati extract exists
Application and whitening articles in whitening articles；Relinqing Granula raw material Herba Polygoni Capitati extract with anti-inflammatory effect；With anti-leaching
The Relinqing Granula raw material Herba Polygoni Capitati extract of coccus effect；For preventing or treating the polygonum capitatum composition of whelk；For
Treat or prevent the polygonum capitatum composition of mouth disease.
Summary of the invention
The object of the present invention is to provide flavonoid glycoside active component in a kind of polygonum capitatum, which mainly includes five flavonoid glycosides
Compound, the polygonum capitatum flavonoid glycoside active component are prepared by following steps:
(1) it crushes and extracts:
After polygonum capitatum is crushed, is extracted at room temperature 2 times with technical grade methanol solution, 1 day every time, filter concentration, obtain first
Alcoholic extract study is stood with ethyl acetate and water extraction and separation, after ethyl acetate layer reduced pressure is spin-dried for, obtains acetic acid
Methacrylate layer study；
(2) it separates and purifies:
By ethyl acetate layer study, first through silica gel opening post separation, (200-300 mesh, solvent system are n-hexane: dichloromethane
Alkane, methylene chloride: ethyl acetate and methanol), being further open by octadecyl silane, (solvent system is post separation
Methanol: water), obtaining two flavonoid glycoside active components, (eluent system is respectively that 40% methanol aqueous solution and 50% methanol are water-soluble
Liquid), two active components again respectively through octadecyl silane be open post separation (solvent system is methanol: water), obtain compared with
Pure flavonoid glycoside active component, is then purified with reversed-phase HPLC respectively again, and mobile phase is methanol aqueous solution, finally obtains five Huangs
Glycoside compounds；Five compounds are myricetin 7-O- α-L-rhamnopyranoside, quercetin 3-O- respectively
β-D-glucopyranoside, Folecitin, quercetin 3-O- α-(2 "-galloyl) rhamnoside, and
kaempferol 3-O-α-L-rhamnopyranoside。
N-hexane in the step (2): dichloromethane solvent system is by volume are as follows: 100: 0,90: 10,70: 30,50:
50,30: 70,10: 90,0: 100；Subsequent methylene chloride: ethyl acetate solvent system is by volume are as follows: 90: 10,70: 30,50:
50,30: 70,0: 100；It is finally methanol.
The methanol of first time octadecyl silane opening column purification in step (2): aqueous solvent system is by volume are as follows:
30: 70,35: 65,40: 60,50: 50,60: 40,70: 30,90: 10,100: 0.
The methanol of second of octadecyl silane opening column purification in step (2): aqueous solvent system divides by volume
Not are as follows: 30: 70,50: 50 and 30: 70,35: 65,60: 40.
The chromatographic column and mobile phase that HPLC is purified in step (2) are respectively CAPCELL PAKC18, methanol: water=38: 62
With Develosil C30-UG-5, methanol: water=48: 52.
It is a further object to provide the flavonoid glycoside active components to prepare answering in anti-phytophthora pesticide
With the flavonoid glycoside active component mainly includes five flavonoid glycoside compounds.The research of the invention finds that this active component and chemical combination
Object can significantly inhibit the generation of phytophthora asexual spore capsule in the screening model of anti-phytophthora compound, to reach suppression
The effect of phytophthora sprawling processed.Drug of the present invention be with flavonoid glycoside active component or five flavonoid glycoside compounds for activity at
Part, it is prepared with pharmaceutically acceptable carrier or excipient.
Pharmaceutical dosage form of the invention selects solid pharmaceutical preparation or liquid preparation.
Polygonum capitatum flavonoid glycoside component provided by the invention and five flavonoid glycoside compounds show to show in Phytophthora capsici
The activity for inhibiting phytophthora asexual spore capsule to generate of work.Preparation method of the present invention is simple, and the product purity of extraction is high, and headdress flower
Knotweed is the cheap Chinese medicine that is easy to get, and the research and development for anti-phytophthora drug provide basic research, is had important practical significance.
Detailed description of the invention
Fig. 1 is the inhibiting effect that flavonoid glycoside mixture A2, B2 generates phytophthora asexual spore capsule.
Fig. 2 is the inhibiting effect that five flavonoid glycoside compounds generate phytophthora asexual spore capsule.1:myricetin in figure
7-O- α-L-rhamnopyranoside, 2:quercetin 3-O- β-D-glucopyranoside, 3:Folecitin, 4:
Quercetin 3-O- α-(2 "-galloyl) rhamnoside, 5:kaempferol 3-O- α-L-rhamnopyranoside.
Fig. 3 is the displaing micro picture for the generation that five flavonoid glycoside compounds inhibit phytophthora asexual spore capsule.A is feminine gender in figure
Control: 0.2%DMSO, b are positive control: metalaxyl (30 μ g/mL), and c is myricetin 7-O- α-L-
Rhamnopyranoside (30 μM), d are quercetin 3-O- β-D-glucopyranoside (30 μM), and e is
Folecitin (3 μM), f are quercetin 3-O- α-(2 "-galloyl) rhamnoside (3 μM), g kaempferol
3-O-α-L-rhamnopyranoside(10μM)。
Specific embodiment
Below in conjunction with drawings and examples, the present invention is further illustrated.But it is above-mentioned that this should not be interpreted as to the present invention
The range of theme is only limitted to following examples, and all technologies realized based on above content of the present invention belong to model of the invention
It encloses.
Embodiment 1 separation and Extraction flavonoid glycoside component and preparation method of five flavonoid glycoside compounds from polygonum capitatum
(1) it crushes and extracts:
After the polygonum capitatum for taking 250g dry crushes, is extracted 2 times, 1 day every time, filtered at room temperature with 2.5L methanol (technical grade)
Concentration, obtains methanol extract study, with ethyl acetate and water extraction and separation, stands, after ethyl acetate layer reduced pressure is spin-dried for,
Obtain ethyl acetate layer study 9g.
(2) it separates and purifies:
By ethyl acetate layer study first through silica gel opening post separation (200-300 mesh, following solvent system by volume, just
Hexane: dichloromethane solvent system is by volume are as follows: 100: 0,90: 10,70: 30,50: 50,30: 70,10: 90,0: 100；With
Methylene chloride afterwards: ethyl acetate solvent system is by volume are as follows: 90: 10,70: 30,50: 50,30: 70,0: 100；It is finally first
Alcohol)；By active sample again with ODS opening post separation (solvent system is methanol by volume: water=0: 70,35: 65,40: 60,
50: 50,60: 40,70: 30,90: 10,100: 0)；Obtain two flavonoid glycoside mixture A1 (975.7mg) and B1
(267.4mg), then A1 and B1 respectively with ODS opening post separation (solvent system by volume be respectively methanol: water are as follows: 30:
70,50: 50 and 30: 70,35: 65,60: 40), obtaining the higher flavonoid glycoside mixture A2 (129.4mg) of purity and B2
(139.9mg).A2 50mg is taken to be purified with HPLC, chromatographic condition: chromatographic column PAKC18(10/250mm), flow velocity 3ml/min, detection
Wavelength 210nm, mobile phase are methanol: water=38: 62, obtain compound myricetin 7-O- α-L-
Rhamnopyranoside (2.7mg, retention time 19.5min), quercetin 3-O- β-D-glucopyranoside
(2.6mg, retention time 25.3min), Folecitin (35.8mg, retention time 38.0min)；Take B2 20mg HPLC
Purifying, chromatographic condition: chromatographic column C30-UG-5 (10/250mm), flow velocity 3ml/min, Detection wavelength 210nm, mobile phase are first
Alcohol: water=48: 52, obtain compound quercetin 3-O- α-(2 "-galloyl) rhamnoside (5.3mg, retention time
For 54.8min), kaempferol 3-O- α-L-rhamnopyranoside (2.5mg, retention time 60.6min).Five
The structural formula of compound is as follows:
Embodiment 2
To 1 gained compound myricetin 7-O- α-L-rhamnopyranoside, quercetin 3-O- of embodiment
β-D-glucopyranoside, Folecitin, quercetin 3-O- α-(2 "-galloyl) rhamnoside, and
The physicochemical characteristics of kaempferol 3-O- α-L-rhamnopyranoside and the Qualitative Identification of chemical structure:
Compound myricetin 7-O- α-L-rhamnopyranoside, quercetin 3-O- β-D-
Glucopyranoside, Folecitin, quercetin 3-O- α-(2 "-galloyl) rhamnoside, and kaempferol
The structure of 3-O- α-L-rhamnopyranoside is compared through 1H NMR, MS and document and is determined
The physicochemical property of compound myricetin 7-O- α-L-rhamnopyranoside: yellow-brown solid, molecular formula
For C21H20O12；ESI-TOF-MS:m/z 465.1027 (M+H)+, hydrogen modal data:1H NMR(500MHz,CD3OD): δ=6.95
(s, 2H), 6.37 (d, 1H, J=2.0Hz), 6.20 (d, 1H, J=2.0Hz), 5.31 (d, 1H, J=1.5Hz), 4.22 (dd,
1H, J=3,1.5Hz), 3.79 (dd, 1H, J=9.0,3.0Hz), 3.52 (m, 1H), 3.32 (m, 1H), 0.96 (d, 3H, J=
6.5Hz)。
The physicochemical property of compound quercetin 3-O- β-D-glucopyranoside: yellow-brown solid, molecular formula are
C21H20O12；ESI-TOF-MS:m/z 465.1027 (M+H)+, hydrogen modal data:1H NMR(500MHz,CD3OD): δ=7.70 (d,
1H, J=2.0Hz), 7.58 (dd, 1H, J=8.5,2.0Hz), 6.86 (d, 1H, J=8.5Hz), 6.19 (d, 1H, J=
2.0Hz), 6.38 (d, 1H, J=2.0Hz), 5.22 (d, 1H, J=8.0Hz), 3.47-3.20 (m, 4H), 3.70 (dd, 1H, J=
), 12.0,2.5Hz 3.57 (dd, 1H, J=13.0,6.0Hz).
The physicochemical property of compound Folecitin: yellow-brown solid, molecular formula C21H20O11；ESI-TOF-MS:m/z
449.1114(M+H)+, hydrogen modal data:1H NMR(500MHz,CD3OD): δ=7.33 (d, 1H, J=1.5Hz), 7.30 (dd,
1H, J=8.0,1.5Hz), 6.90 (d, 1H, J=8.0Hz), 6.18 (d, 1H, J=2.0Hz), 6.35 (d, 1H, J=2.0Hz),
5.34 (s, 1H), 4.21 (s, 1H), 3.39 (m, 1H), 3.73 (dd, 1H, J=9.5,3.0Hz), 3.43 (m, 1H), 0.93 (d,
3H, J=6.0Hz).
Compound quercetin 3-O- α-(2 "-galloyl) rhamnoside physicochemical property: yellow solid, molecule
Formula is C28H24O15；ESI-TOF-MS:m/z 601.1179 (M+H)+, hydrogen modal data:1H NMR(500MHz,CD3OD): δ=
7.36 (d, 1H, J=2.0Hz), 7.34 (dd, 1H, J=8.5,2.0Hz), 7.07 (s, 2H), 6.93 (d, 1H, J=85Hz),
6.18 (d, 1H, J=2.0Hz), 6.36 (d, 1H, J=2.0Hz), 5.63 (dd, 1H, J=3.5,2.0Hz), 5.50 (d, 1H, J
=1.5Hz), 4.01 (m, 1H), 3.47 (m, 2H), 1.03 (d, 3H, J=6.0Hz).
The physicochemical property of compound kaempferol 3-O- α-L-rhamnopyranoside: yellow-brown solid, molecular formula
For C21H20O10；ESI-TOF-MS:m/z 443.1125 (M+H)+, hydrogen modal data:1H NMR(500MHz,CD3OD): δ=7.77
(d, 1H, J=8.5Hz), 6.93 (d, 1H, J=8.5Hz), 6.18 (d, 1H, J=2.0Hz), 6.35 (d, 1H, J=2.0Hz),
5.37 (d, 1H, J=1.5Hz), 4.21 (dd, 1H, J=3.0,1.5Hz), 3.70 (dd, 1H, J=9.0,3.5Hz), 3.33-
3.16 (m, 2H), 0.92 (d, 3H, J=5.5Hz).
Embodiment 3
It is asexual to significantly inhibit phytophthora in anti-phytophthora screening model for flavonoid glycoside component and five flavonoid glycoside compounds
The generation of sporangium.
Current anti-phytophthora active matter Quality Research, which is concentrated mainly on, inhibits mycelia growth and zoospore to sprout two aspects,
Inhibit the active matter Quality Research of phytophthora asexual reproduction phases sporangium generation few to having.Metalaxyl is to prevent and treat at present
It, can the mycelial production grown with asexual spore capsule of strong inhibition phytophthora by a kind of most widely used pesticide above vegetable plague
It is raw.Present invention discover that polygonum capitatum flavonoid glycoside mixture and five flavonoid glycoside compounds are in Phytophthora capsici model, significant phytophthora
The generation of bacterium asexual spore capsule.
1. experimental method:
(1) Phytophthora capsici bacterial strain is taken out from 10 DEG C of biochemical cultivation cases, recovers to 25 DEG C, a fritter is taken to be inoculated into 20ml solid
In culture dish (10% V8juice, 0.02% calcium carbonate, 2% agar powder).25 DEG C be protected from light culture 3-4d it is spare.
(2) it takes V8 juice 50mL, calcium carbonate 0.2g to mix in centrifuge tube, is centrifuged 15min (4000r/min), obtains
To V8 juice supernatant.
(3) solid plate for being protected from light culture 3-4d in biochemical cultivation case is taken out, is cut directly with punch at mycelia edge
Diameter is the fungus block of 6mm, is placed on the culture dish center that sterilized diameter is 6cm, and 10mL fluid nutrient medium (3% is then added
V8 juice supernatant), it is put in 25 DEG C of biochemical cultivation cases and is protected from light culture.After 48h, remove culture medium, and be added 10mL sterile water/
Ware.It when sample is added, does not first use 20 μ L DMSO to dissolve in sample, then draws the sterile water 980L in culture dish with liquid-transfering gun,
It is added in sample solution, is even added in culture dish again after mixing.Using 0.2%DMSO as negative control in measurement, with 30 μ
The metalaxyl of g/mL is positive control.It is protected from light culture by 16-24h, observes phytophthora asexual spore under an optical microscope
The case where capsule generates and the counting that suspends (10 times of eyepieces, 4 times of object lens) statistics, then calculate its inhibiting rate.
Inhibiting rate (%)=(ND-NS)/NC× 100%
ND: the asexual spore capsule number generated in negative control group unit volume；NS: the nothing generated in sample sets unit volume
Spermatium capsule number.
2. experimental result:
The inhibiting rate point that flavonoid glycoside mixture A2 and B2 under 3 μ g/mL concentration generates phytophthora asexual spore capsule
It Wei not 88% and 98% (Fig. 1).Five flavonoid glycoside compounds are respectively to epidemic disease under 30 μM, 30 μM, 3 μM, 3 μM, 10 μM of concentration
The inhibiting rate that mould asexual spore capsule generates is 74%, 65%, 86%, 78%, 90% (Fig. 2) and corresponding displaing micro picture (figure
3)。
Claims (3)
1. flavonoid glycoside active component in a kind of polygonum capitatum, which is characterized in that in the polygonum capitatum flavonoid glycoside active component by with
Lower step prepares:
(1) it crushes and extracts:
After polygonum capitatum is crushed, is extracted at room temperature 2 times with technical grade methanol solution, 1 day every time, filter concentration, obtain methanol leaching
Extract study is stood with ethyl acetate and water extraction and separation, after ethyl acetate layer reduced pressure is spin-dried for, obtains ethyl acetate
Layer study；
(2) it separates and purifies:
By ethyl acetate layer study first through silica gel opening post separation, separation condition: 200-300 mesh, solvent system are n-hexane: two
Chloromethanes, methylene chloride: then ethyl acetate and methanol pass through first time octadecyl silane opening post separation, solvent
System is methanol: water, and it is living that two flavonoid glycosides are obtained when eluent system is respectively 40% methanol aqueous solution and 50% methanol aqueous solution
Property component, two flavonoid glycoside active components are open post separation through second octadecyl silane respectively again, and solvent system is
Methanol: water obtains purer flavonoid glycoside active component, is then purified respectively with reversed-phase HPLC again, and mobile phase is methanol aqueous solution,
Finally obtain five flavonoid glycoside compounds；Five compounds are myricetin 7-O- α-L-rhamnopyranoside respectively,
quercetin 3-O-β -D-glucopyranoside, Folecitin, quercetin 3-O-α -(2''-galloyl)
Rhamnoside, and kaempferol 3-O- α-L-rhamnopyranoside；
Wherein n-hexane in the step (2): the volume ratio of dichloromethane solvent system are as follows: 100: 0,90: 10,70: 30,50:
50,30: 70,10: 90,0: 100；Subsequent methylene chloride: ethyl acetate solvent system is by volume are as follows: 90: 10,70: 30,50:
50,30: 70,0: 100；It is finally methanol；
The methanol of first time octadecyl silane opening column purification in the step (2): aqueous solvent system is by volume are as follows:
30: 70,35: 65,40: 60,50: 50,60: 40,70: 30,90: 10,100: 0；
The methanol of second of octadecyl silane opening column purification in the step (2): aqueous solvent system divides by volume
Not are as follows: 30: 70,50: 50 and 30: 70,35: 65,60: 40；
The chromatographic column and mobile phase purified in the step (2) with reversed-phase HPLC are respectively CAPCELL PAKC18, methanol: water=
38: 62 and Develosil C30-UG-5, methanol: water=48: 52；The former obtains compound myricetin 7-O- α-L-
Rhamnopyranoside, quercetin 3-O- β-D-glucopyranoside, Folecitin, the latter obtain compound
Quercetin 3-O- α-(2''-galloyl) rhamnoside and kaempferol 3-O- α-L-
rhamnopyranoside。
2. flavonoid glycoside active component is preparing answering in anti-phytophthora pesticide in a kind of polygonum capitatum according to claim 1
With the flavonoid glycoside active component mainly includes five flavonoid glycoside compounds, which is characterized in that the drug is living with flavonoid glycoside
Property component or five flavonoid glycoside compounds be active ingredient, be made with pharmaceutically acceptable carrier or excipient.
3. application according to claim 2, which is characterized in that the pharmaceutical dosage form selects solid pharmaceutical preparation or liquid preparation.
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