CN105924479B - Kalimeris active component and compound and preparation and application - Google Patents
Kalimeris active component and compound and preparation and application Download PDFInfo
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- CN105924479B CN105924479B CN201610463306.XA CN201610463306A CN105924479B CN 105924479 B CN105924479 B CN 105924479B CN 201610463306 A CN201610463306 A CN 201610463306A CN 105924479 B CN105924479 B CN 105924479B
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- kalimeris
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- phytophthora
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- 241001252483 Kalimeris Species 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 150000001875 compounds Chemical class 0.000 title description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 38
- 241000233614 Phytophthora Species 0.000 claims abstract description 22
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229930182486 flavonoid glycoside Natural products 0.000 claims abstract description 16
- -1 flavonoid glycoside compounds Chemical class 0.000 claims abstract description 15
- 239000000575 pesticide Substances 0.000 claims abstract description 15
- 239000002904 solvent Substances 0.000 claims abstract description 11
- 238000000926 separation method Methods 0.000 claims abstract description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000000741 silica gel Substances 0.000 claims abstract description 6
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- KEIZXGINFPDITQ-UHFFFAOYSA-N UNPD138008 Natural products C1=C(O)C(OC)=CC=C1C1=C(OC2C(C(O)C(O)C(COC3C(C(O)C(O)C(C)O3)O)O2)O)C(=O)C2=C(O)C=C(O)C=C2O1 KEIZXGINFPDITQ-UHFFFAOYSA-N 0.000 claims description 11
- UIDGLYUNOUKLBM-GEBJFKNCSA-N isorhamnetin-3-O-rutinoside Chemical compound C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO[C@H]3[C@@H]([C@H](O)[C@@H](O)[C@H](C)O3)O)O2)O)=C1 UIDGLYUNOUKLBM-GEBJFKNCSA-N 0.000 claims description 11
- BGLPQQKZNUKSAR-UHFFFAOYSA-N isorhamnetin-3-O-rutinoside Natural products COc1ccc(cc1O)C2=C(OC3OC(COCC4OC(O)C(O)C(O)C4O)C(O)C(O)C3O)C(=O)c5c(O)cc(O)cc5O2 BGLPQQKZNUKSAR-UHFFFAOYSA-N 0.000 claims description 11
- IEPKWJCBNGNVDF-UHFFFAOYSA-N narcissin Natural products OC1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)OC2C(C(O)C(O)C(COC3C(C(O)C(O)C(C)O3)O)O2)O)=C1 IEPKWJCBNGNVDF-UHFFFAOYSA-N 0.000 claims description 11
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims description 2
- 239000000839 emulsion Substances 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims 1
- 229930003944 flavone Natural products 0.000 claims 1
- 150000002213 flavones Chemical class 0.000 claims 1
- 235000011949 flavones Nutrition 0.000 claims 1
- 238000005858 glycosidation reaction Methods 0.000 claims 1
- 229930182470 glycoside Natural products 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 13
- 230000002401 inhibitory effect Effects 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 4
- 235000013311 vegetables Nutrition 0.000 abstract description 3
- 230000000975 bioactive effect Effects 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 abstract description 2
- 238000003808 methanol extraction Methods 0.000 abstract 1
- JMGZEFIQIZZSBH-UHFFFAOYSA-N Bioquercetin Natural products CC1OC(OCC(O)C2OC(OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5)C(O)C2O)C(O)C(O)C1O JMGZEFIQIZZSBH-UHFFFAOYSA-N 0.000 description 12
- RTATXGUCZHCSNG-TYSPDFDMSA-N Kaempferol-3-O-rutinoside Natural products OC1[C@H](O)[C@@H](O)C(C)O[C@H]1OCC1[C@@H](O)[C@@H](O)C(O)[C@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=CC(O)=CC=2)=O)O1 RTATXGUCZHCSNG-TYSPDFDMSA-N 0.000 description 12
- RTATXGUCZHCSNG-QHWHWDPRSA-N Nicotiflorin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=CC(O)=CC=2)=O)O1 RTATXGUCZHCSNG-QHWHWDPRSA-N 0.000 description 12
- RTATXGUCZHCSNG-ZFDPGQBLSA-N nicotiflorin Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](OC2=C(c3ccc(O)cc3)Oc3c(c(O)cc(O)c3)C2=O)O1)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](C)O1 RTATXGUCZHCSNG-ZFDPGQBLSA-N 0.000 description 12
- RTATXGUCZHCSNG-UHFFFAOYSA-N nicotiflorine Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=CC(O)=CC=2)=O)O1 RTATXGUCZHCSNG-UHFFFAOYSA-N 0.000 description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 239000002775 capsule Substances 0.000 description 6
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 4
- OVVGHDNPYGTYIT-ROUHPGRKSA-N alpha-L-Rhap-(1->6)-D-Glcp Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 OVVGHDNPYGTYIT-ROUHPGRKSA-N 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- 239000005807 Metalaxyl Substances 0.000 description 3
- 241000233616 Phytophthora capsici Species 0.000 description 3
- 230000011681 asexual reproduction Effects 0.000 description 3
- 238000013465 asexual reproduction Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 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
- 239000013642 negative control Substances 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 241000233622 Phytophthora infestans Species 0.000 description 2
- 239000011149 active material Substances 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- 238000001425 electrospray ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 150000007955 flavonoid glycosides Chemical class 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000014347 soups Nutrition 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241001008148 Aster indicus Species 0.000 description 1
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- 241000723346 Cinnamomum camphora Species 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 244000166124 Eucalyptus globulus Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 208000034507 Haematemesis Diseases 0.000 description 1
- 208000005647 Mumps Diseases 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 206010000269 abscess Diseases 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000003810 ethyl acetate extraction Methods 0.000 description 1
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000014617 hemorrhoid Diseases 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000014759 maintenance of location 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
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 208000010805 mumps infectious disease Diseases 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 201000005737 orchitis Diseases 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Saccharide Compounds (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The present invention provides a kind of kalimeris water soluble active component and two flavonoid glycoside compounds, after Chinese medicine kalimeris is crushed, methanol extraction, filter concentration, after water and ethyl acetate solvent distribution, water layer concentration is first open post separation through ODS, again by the isolated kalimeris active component of silica gel opening column, repurity obtains two flavonoid glycoside compounds.The present invention has found that kalimeris active component and two of them flavonoid glycoside compound can significantly inhibit the generation of phytophthora sporangium by the bioactive systems for inhibiting phytophthora sporangium to generate, and plays anti-phytophthora effect, can apply in preparing anti-phytophthora pesticide.Preparation method of the present invention is simple, and the product purity of extraction is high, and kalimeris is cheap and easily-available vegetables and Chinese medicine, extends the medicinal usage of kalimeris, provides foundation to develop innovation pesticide using traditional Chinese medicine.
Description
Technical field
The invention belongs to pharmaceutical technology field, be related to kalimeris water soluble active component and compound and preparation method and
Prepare the application in resisting phytophthora disease pesticide.
Background technique
Currently, phytophthora alreadys exceed 100 kinds, and increasing month by month, most destructive plant in the world can be caused
Disease all brings serious economic loss every year.In the middle of the 19th century, the late blight of potato caused by phytophthora infestans results in Ai Er
The generation that orchid is hard up greatly ultimately causes more than 100 ten thousand people death, the human world tragedy that more than 100 ten thousand people leave the native place.Drawn by camphor tree phytophthora
The eucalyptus root rot risen makes the forest structure of Australian southwest and plant population that great change have occurred, this is serious
Threaten local bio-diversity and natural ecosystems.Chemical pesticide is long-term and is widely applied and also produces serious drug resistance
Property and environmental pollution.Therefore, find and find that the efficient anti-phytophthora active material of low toxicity has become urgent problem.
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, and the generation of sporangium is epidemic disease
The critical period of mould asexual reproduction phases, the zoospore generated are the bases of phytophthora sprawling.Therefore finding has suppression
The active material that asexual spore capsule processed generates has and its important meaning.
Natural products possesses materials naturally as drug, from a wealth of sources；Effect is steady；Toxic side effect is relatively small；It is medicine
The feature of the first-class of the best source of object primer, thus be widely studied.In addition, China's natural resources of Chinese medicinal materials very abundant, very much
Chinese medicine is owned by long applicating history, therefore finding from Chinese medicine, there is the active ingredient of anti-phytophthora to have biggish advantage.
Kalimeris (Kalimeris indica) belongs to composite family kalimeris category, herbaceos perennial also known as roadside chrysanthemum.At me
State, as Chinese medicine for treating flu, diarrhea, gastric ulcer, acute abscess gastric, gastroenteritis, acute orchitis, haematemesis and damage
Etc. diseases.Main chemical compositions have phenols, terpene, polysaccharide, volatile oil etc..With numerous bioactivity, ground extensively always
Study carefully.Such as: kalimeris is preparing the application in blood lipid-lowering medicine or functional food；The siberian cocklour and Indian kalimeris herb for treating mumps
Soup；A kind of kalimeris soup etc. for treating hemorrhoid.
Summary of the invention
It is described the object of the present invention is to provide a kind of kalimeris water soluble active component and two of them flavonoid glycoside compounds
Kalimeris water soluble active component and two of them flavonoid glycoside compounds are realized by following preparation step:
(1) kalimeris is extracted with methanol after crushed, and suction filtration is distributed through water and ethyl acetate solvent again after being concentrated, water layer is dense
Contracting, obtains extract；
(2) gained extract is obtained kalimeris and phytophthora sporangium is inhibited to generate successively through ODS and silica gel opening post separation
Active component；
(3) gained kalimeris active component obtains two flavonoid glycoside compounds: kaempferol-3-O- through HPLC after purification
Rutinoside and isorhamnetin-3-O-rutinoside.
Kalimeris used is dry kalimeris complete stool in step (1).
The solvent system of ODS (octadecylsilane chemically bonded silica filler) opening post separation is methanol: water in step (2):
Solvent system is methanol by volume: water=3:7,5:5,6:4,7:3,8:2,9:1,10:0.
The solvent system of silica gel opening post separation is ethyl acetate: methanol=7:3,5:5,3:7,0:10 in step (2).
The chromatographic condition that HPLC is purified in step (3): chromatographic column C18 (Φ 10/250mm), flow velocity 3mL/min detect wave
Long 210nm, mobile phase are methanol: water=35: 65.
Two flavonoid glycoside compounds kaempferol-3-O-rutinoside and isorhamnetin-3-O-
Content of the rutinoside in kalimeris water soluble active component is between 51%~72%.Structural formula is as follows:
It is a further object to provide the kalimeris water soluble active components and two flavonoid glycoside compounds to exist
Prepare the application in anti-phytophthora pesticide.The pesticide is by kalimeris water soluble active component and two flavonoid glycoside compounds and pesticide
Acceptable carrier composition on.The present invention is research shows that kalimeris water soluble active component and two of them flavonoid glycoside compounds can
To inhibit the generation of phytophthora sporangium significantly.
Pharmaceutical dosage form of the invention selects pulvis, emulsion or granule etc..
The present invention has found kalimeris active component and two of them by the bioactive systems for inhibiting phytophthora sporangium to generate
Flavonoid glycoside compound can significantly inhibit the generation of phytophthora sporangium, play anti-phytophthora effect, can prepare anti-phytophthora pesticide
Middle application.Preparation method of the present invention is simple, and the product purity of extraction is high, and kalimeris is cheap and easily-available vegetables and Chinese medicine, extension
The medicinal usage of kalimeris provides reference to develop innovation pesticide using traditional Chinese medicine.Preparation method of the present invention is simple, mentions
The product purity taken is high, and kalimeris is cheap and easily-available Chinese medicine, provides foundation to develop innovation pesticide using traditional Chinese medicine.
Detailed description of the invention
Fig. 1 is the inhibiting rate that kalimeris water soluble active component generates phytophthora sporangium.
Fig. 2 is compound kaempferol-3-O-rutinoside and isorhamnetin-3-O-rutinoside to epidemic disease
The inhibiting rate that mycotic spore capsule generates, wherein left: kaempferol-3-O-rutinoside, it is right: isorhamnetin-3-O-
rutinoside。
Fig. 3 is that compound kaempferol-3-O-rutinoside and isorhamnetin-3-O-rutinoside inhibit
The displaing micro picture that phytophthora sporangium generates.A is negative control in figure: 0.2%DMSO, b are positive control: metalaxyl (30 μ g/
ML), c is kaempferol-3-O-rutinoside (30 μM) d, kaempferol-3-O-rutinoside (30 μM).
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.
1 kalimeris water soluble active component of embodiment and two of them flavonoid glycoside compounds kaempferol-3-O-
The preparation method of rutinoside and isorhamnetin-3-O-rutinoside
(1) it crushes and extracts:
After the kalimeris for taking 1kg dry crushes, is extracted 2 times, 1 day every time, filtered dense at room temperature with 10L methanol (technical grade)
Contracting, obtains methanol extract study, with ethyl acetate and water extraction and separation, stands, and after water layer reduced pressure is spin-dried for, obtains second water
Layer study 28g.
(2) it separates and purifies:
By water layer study first through ODS opening post separation (solvent system is methanol by volume: water=3:7,5:5,6:4,7:
3,8:2,9:1,10:0)；Active sample use again silica gel opening post separation (200-300 mesh, following solvent system by volume, second
Acetoacetic ester: methanol=7:3,5:5,3:7,0:10)；Obtain the active component (152mg) containing reactive compound of yellow.It takes
17.4mg active component is purified with reverse hplc, chromatographic condition: chromatographic column C18 (Φ 10/250mm), flow velocity 3ml/min, detection
Wavelength 210nm, mobile phase are methanol: water=35: 65, obtain kaempferol-3-O-rutinoside (4.1mg, when reservation
Between be 60.0min) and isorhamnetin-3-O-rutinoside (4.8mg, retention time 69.7min).
Embodiment 2
To embodiment 1 gained compound kaempferol-3-O-rutinoside and isorhamnetin-3-O-
The physicochemical characteristics of rutinoside and the Qualitative Identification of chemical structure:
The structure of compound kaempferol-3-O-rutinoside and isorhamnetin-3-O-rutinoside pass through1H NMR, MS and document, which compare, to be determined
The physicochemical property of compound kaempferol-3-O-rutinoside: yellow solid,Molecular formula is C27H30O15；ESI-TOF-MS:m/z 595.1663(M+H)+, hydrogen modal data:1H NMR(500MHz,CD3OD): δ=1.12 (3H, d, J=6.0Hz, H-6 " '), 3.25-2.81 (10H, m, H-2 " -6 "/2 " ' -
5 " '), 4.51 (1H, s, H-1 " '), 5.12 (1H, d, J=7.5Hz, H-1 "), 6.89 (2H, d, J=8.5Hz, H-3 '/5 '),
8.07 (2H, d, J=8.5Hz, H-2 '/6 '), 6.41 (1H, s, H-8), 6.21 (1H, s, H-6).
The physicochemical property of compound isorhamnetin-3-O-rutinoside: yellow solid,Molecular formula is C28H32O16,ESI-TOF-MS:m/z 625.1765(M+H)+, hydrogen spectrum number
According to:1H NMR(500MHz,CD3OD): δ=1.09 (3H, d, J=6.0Hz, H-6 " '), 3.22-3.49 (8H, m, H-2 " -5 "/
- 5 " '), 2 " ' 3.60 (1H, dd, J=3.5,2.0Hz, H-6b "), 3.81 (1H, dd, J=12.5,1.5Hz, H-6a "), 4.52
(1H, d, J=1.5Hz, H-1 " '), 5.24 (1H, d, J=7.0Hz, H-1 "), 7.62 (1H, dd, J=8.5,2.0Hz, H-6 '),
6.91 (H, d, J=8.5Hz, H-5 '), 7.94 (1H, d, J=2.0Hz, H-2 '), 3.94 (3H, s ,-OCH3),6.40(1H,d,J
=2.0Hz), 6.20 (1H, d, J=2.0Hz).
Embodiment 3
Kalimeris water soluble active component and compound can significantly inhibit phytophthora sporangium in Phytophthora capsici screening model
Generation.
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 kalimeris water soluble active component and two of them flavonoid glycoside compounds kaempferol-3-O-
Rutinoside and isorhamnetin-3-O-rutinoside significantly inhibits phytophthora sporangium in Phytophthora capsici model
Generation.
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 V8juice 50mL, calcium carbonate 0.2g to mix in centrifuge tube, is centrifuged 15min (4000r/min), obtains
V8juice 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, 980 μ of sterile water in culture dish is then drawn with liquid-transfering gun
L is added in sample solution, is even added in culture dish again after mixing.Using 0.2%DMSO as negative control in measurement, with
The metalaxyl of 30 μ g/mL is positive control.It is protected from light culture by 16-24h, observes the asexual spore of phytophthora under an optical microscope
The case where ascus 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)/ND× 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:
Kalimeris active component significantly inhibits (Fig. 1) to what phytophthora sporangium generated in 30 μ g/mL.Chemical combination
Object kaempferol-3-O-rutinoside and isorhamnetin-3-O-rutinoside is in 3-30 μM of concentration range
(Fig. 2) inhibited to the generation of phytophthora sporangium.Both compounds are when concentration is 30 μM, to phytophthora spore
Capsule inhibiting rate is respectively 92% and 83%, corresponding displaing micro picture (Fig. 3).
Claims (5)
1. a kind of kalimeris water soluble active component, which is characterized in that the kalimeris water soluble active component and two flavones glycosidations
Object is closed to be prepared by the following:
(1) kalimeris is extracted with methanol after crushed, is concentrated, is obtained through water and ethyl acetate solvent distribution, water layer again after filtering concentration
To water layer study；
(2) gained water layer study is obtained into the work that kalimeris inhibits phytophthora sporangium to generate successively through ODS and silica gel opening post separation
Property component；Wherein the solvent system of ODS opening post separation is methanol: water, solvent system are methanol by volume: water=3:7,5:
5, 6:4, 7:3, 8:2, 9:1, 10:0；The solvent system of silica gel opening post separation is ethyl acetate: methanol, volume ratio are
7:3, 5:5, 3:7, 0:10；
(3) gained kalimeris active component obtains two flavonoid glycoside compounds: kaempferol-3- through HPLC after purificationO-
Rutinoside and isorhamnetin-3-O- rutinoside, structural formula is as follows, and two flavonoid glycoside compounds are water-soluble in kalimeris
Property active component in content between 51% ~ 72%, HPLC purifying chromatographic condition are as follows: chromatographic column C18 Φ 10/250mm, stream
3 mL/min of speed, 210 nm of Detection wavelength, mobile phase is methanol: water=35: 65,
。
2. a kind of kalimeris water soluble active component according to claim 1, which is characterized in that kalimeris used in step (1)
For dry kalimeris complete stool.
3. kalimeris water soluble active component described in claim 1 and two flavonoid glycoside compounds are in preparing anti-phytophthora pesticide
Application.
4. application according to claim 3, which is characterized in that the pesticide is by kalimeris water soluble active component and two Huangs
Acceptable carrier forms on glycoside compounds and Pesticide Science.
5. application according to claim 3, which is characterized in that the dosage form of the pesticide selects pulvis, emulsion or granule.
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| WO2014003808A1 (en) * | 2012-06-27 | 2014-01-03 | King Saud University | Method of extracting kaempferol-based antioxidants from solenostemma arghel |
| CN103113436A (en) * | 2012-10-23 | 2013-05-22 | 北京华润高科天然药物有限公司 | Method for preparing kaempferol-3-O-rutinoside from ginkgo leaf extract |
| CN104130299A (en) * | 2014-07-01 | 2014-11-05 | 昆明理工大学 | Extraction separation method for isorhamnetin-3-O-beta-D-rutinoside in caragana sinica flower bud |
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