CN116041408B - Preparation method and application of flavonol compound dysosma versipellis A-F - Google Patents
Preparation method and application of flavonol compound dysosma versipellis A-F Download PDFInfo
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- CN116041408B CN116041408B CN202310045919.1A CN202310045919A CN116041408B CN 116041408 B CN116041408 B CN 116041408B CN 202310045919 A CN202310045919 A CN 202310045919A CN 116041408 B CN116041408 B CN 116041408B
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- 241000332747 Dysosma versipellis Species 0.000 title claims abstract description 71
- HVQAJTFOCKOKIN-UHFFFAOYSA-N flavonol Natural products O1C2=CC=CC=C2C(=O)C(O)=C1C1=CC=CC=C1 HVQAJTFOCKOKIN-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 235000011957 flavonols Nutrition 0.000 title claims abstract description 53
- -1 flavonol compound Chemical class 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title abstract description 15
- 150000002576 ketones Chemical class 0.000 claims abstract description 29
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 7
- 235000006708 antioxidants Nutrition 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 231
- 239000012046 mixed solvent Substances 0.000 claims description 104
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 98
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 89
- 239000000741 silica gel Substances 0.000 claims description 81
- 229910002027 silica gel Inorganic materials 0.000 claims description 81
- 238000010828 elution Methods 0.000 claims description 79
- 238000004809 thin layer chromatography Methods 0.000 claims description 77
- 238000004440 column chromatography Methods 0.000 claims description 71
- 238000001514 detection method Methods 0.000 claims description 68
- 238000004458 analytical method Methods 0.000 claims description 62
- 230000014759 maintenance of location Effects 0.000 claims description 60
- 238000002953 preparative HPLC Methods 0.000 claims description 59
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 51
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 48
- 150000001875 compounds Chemical class 0.000 claims description 40
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 38
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 36
- 239000012156 elution solvent Substances 0.000 claims description 32
- 239000000499 gel Substances 0.000 claims description 24
- 238000000605 extraction Methods 0.000 claims description 23
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 22
- 239000000284 extract Substances 0.000 claims description 22
- 239000000469 ethanolic extract Substances 0.000 claims description 21
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 20
- 239000002904 solvent Substances 0.000 claims description 20
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- HWJHWSBFPPPIPD-UHFFFAOYSA-N ethoxyethane;propan-2-one Chemical compound CC(C)=O.CCOCC HWJHWSBFPPPIPD-UHFFFAOYSA-N 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 9
- 239000003208 petroleum Substances 0.000 claims description 9
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- ROAYSRAUMPWBQX-UHFFFAOYSA-N ethanol;sulfuric acid Chemical compound CCO.OS(O)(=O)=O ROAYSRAUMPWBQX-UHFFFAOYSA-N 0.000 claims description 5
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- FUKUFMFMCZIRNT-UHFFFAOYSA-N hydron;methanol;chloride Chemical compound Cl.OC FUKUFMFMCZIRNT-UHFFFAOYSA-N 0.000 claims 1
- 238000004811 liquid chromatography Methods 0.000 claims 1
- 238000005481 NMR spectroscopy Methods 0.000 description 25
- 229910052799 carbon Inorganic materials 0.000 description 23
- 150000007946 flavonol Chemical class 0.000 description 23
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 18
- 229910052739 hydrogen Inorganic materials 0.000 description 18
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- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 13
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 13
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 12
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 12
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 10
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 9
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 9
- 150000002216 flavonol derivatives Chemical class 0.000 description 8
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 6
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- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 5
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- 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 4
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- 230000036542 oxidative stress Effects 0.000 description 3
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- 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 2
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 2
- KZNIFHPLKGYRTM-UHFFFAOYSA-N apigenin Chemical compound C1=CC(O)=CC=C1C1=CC(=O)C2=C(O)C=C(O)C=C2O1 KZNIFHPLKGYRTM-UHFFFAOYSA-N 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- AEMOLEFTQBMNLQ-QIUUJYRFSA-N beta-D-glucuronic acid Chemical group O[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-QIUUJYRFSA-N 0.000 description 2
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- 229930003935 flavonoid Natural products 0.000 description 2
- 235000017173 flavonoids Nutrition 0.000 description 2
- 230000007760 free radical scavenging Effects 0.000 description 2
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 2
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- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 2
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- 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 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
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- 238000001228 spectrum Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- XILIYVSXLSWUAI-UHFFFAOYSA-N 2-(diethylamino)ethyl n'-phenylcarbamimidothioate;dihydrobromide Chemical compound Br.Br.CCN(CC)CCSC(N)=NC1=CC=CC=C1 XILIYVSXLSWUAI-UHFFFAOYSA-N 0.000 description 1
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- 206010019468 Hemiplegia Diseases 0.000 description 1
- 208000032982 Hemorrhagic Fever with Renal Syndrome Diseases 0.000 description 1
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- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 1
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- VILAVOFMIJHSJA-UHFFFAOYSA-N dicarbon monoxide Chemical group [C]=C=O VILAVOFMIJHSJA-UHFFFAOYSA-N 0.000 description 1
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- UFWIBTONFRDIAS-UHFFFAOYSA-N naphthalene-acid Natural products C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/42—Preservation of non-alcoholic beverages
- A23L2/44—Preservation of non-alcoholic beverages by adding preservatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3544—Organic compounds containing hetero rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
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- 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
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- 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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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to flavonol compounds, a preparation method and application thereof, which can effectively solve the problems of preparation of flavonol compounds and preparation of antioxidants, wherein the flavonol compounds are dysosma versipellis ketone A (dysosmaflavonoid A), dysosma versipellis ketone B (dysosmaflavonoid B), dysosma versipellis ketone C (dysosmaflavonoid C), dysosma versipellis ketone D (dysosmaflavonoid D), dysosma versipellis ketone E (dysosmaflavonoid E) and dysosma versipellis ketone F (dysosmaflavonoid F) extracted from dysosma versipellis.
Description
Technical Field
The invention relates to the field of medicines, in particular to a preparation method and application of flavonol compound dysosma versipellis A-F with antioxidant activity.
Background
Oxidative stress means that active oxygen free radicals generated in tissues or cells of a body exceed the original oxygen free radical utilization capacity or scavenging capacity, and redox balance is destroyed, so that excessive accumulated active oxygen free radicals and related metabolites thereof are generated in the body to cause certain damage to the body, and certain pathological conditions are presented. Oxidative stress is thought to be responsible for many degenerative diseases such as cancer, chronic inflammation, cardiovascular disease, neurological disease, diabetes, alzheimer's disease, parkinson's disease, and the like. Thus, inhibition of oxidative stress-like damage has become one of the important ways to treat diseases associated with oxidative stress. At the same time, in foods, oxidation of nutrients can cause degradation of the food and even disease of the body of the ingester. Thus, the search for safe antioxidants has been a hotspot technology for pharmaceutical and nutritional research.
Dysosma versipellis is the dried rhizome of Dysosma versipellis Dysosma versipellis of berberidaceae, and is mainly distributed in Zhejiang, guangxi, hubei, hunan, sichuan, guizhou, etc. Has effects of clearing heat and detoxicating, eliminating phlegm and resolving masses, and relieving pain and detumescence. Is used for treating traumatic injury, hemiplegia, joint ache, venomous snake bite, sore, carbuncle, toxic swelling, condyloma acuminatum, epidemic hemorrhagic fever, japanese encephalitis, lymphadenitis, parotitis, breast cancer, esophagus cancer and other inflammatory diseases. The dysosma versipellis contains a large amount of aryl naphthalene lactone type lignans and flavonoid compounds, and the flavonoid compounds from natural sources have pharmacological actions of anti-inflammatory, anti-cancer, anti-aging, heart protection, neuroprotection, immunoregulation, anti-diabetes, antibacterial, antiviral, antioxidation, antiparasitic and the like. The invention relates to a preparation method of flavonol compounds dysosma versipellis A-F and application of the flavonol compounds dysosma versipellis A-F in food and beverage in antioxidant biological activity, and has not been reported so far.
Disclosure of Invention
Aiming at the situation, the invention aims to overcome the defects of the prior art and provide a preparation method and application of the flavonol compound dysosma versipellis A-F, which can effectively solve the problems of preparation of flavonol compounds and preparation of antioxidants.
The technical scheme is that the flavonol compound is dysosma versipellis ketone A-F (dysosmaflavonoids A-F) extracted from dysosma versipellis, and the molecular structural formulas are respectively as follows:
the preparation method comprises the following steps:
20-40kg of dysosma versipellis medicinal material is taken as a raw material, 3-5 times of ethanol with the weight volume and the volume concentration of 95% is added each time, the ethanol is heated and refluxed for extraction for 3 times, the weight volume is calculated by kg of solid, the liquid is calculated by L, the extraction temperature is 92-95 ℃, the extraction time is 1-1.5 hours each time, and the ethanol with the volume concentration of 95% is recovered under reduced pressure to obtain an extract of 95% ethanol; adding 3-5 times of ethanol with weight volume and volume concentration of 50% into the residue, reflux extracting for 1 time at 92-95deg.C for 1-1.5 hr, and recovering 50% ethanol under reduced pressure to obtain extract of 50% ethanol; mixing 95% ethanol extract and 50% ethanol extract, adding anhydrous ethanol 2 times the weight of the extract for dissolution, adding diatomite 2 times the weight of the extract for adsorption, recovering solvent, sequentially adding dichloromethane, ethyl acetate and methanol 3 times the weight of the extract for elution, and recovering solvent to obtain dichloromethane elution part, ethyl acetate elution part and methanol elution part respectively; purifying the methanol eluted part with silica gel column Spectrum separation, sequentially carrying out gradient elution by using a dichloromethane-methanol mixed solvent system with volume ratios of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:30, 100:50 and 0:100, wherein each gradient uses 30L-60L eluent, the flow rate is 50-70mL/min, each 5L-10L is a flow, 54 flow parts are collected, each flow part is detected and analyzed by silica gel thin layer chromatography, and GF is used for detecting and analyzing the flow parts by using GF 254 The thin layer plate is prepared by respectively taking petroleum ether-acetone with the volume ratio of 4:5 and methylene dichloride-methanol with the volume ratio of 5:2 as developing agents, taking sulfuric acid-ethanol solution with the volume ratio of 10:90 as a developing agent, heating for 3-5min at the temperature of 105 ℃, and respectively combining 1-12 parts, 13-24 parts, 25-30 parts, 31-36 parts, 37-38 parts, 39-42 parts, 43-44 parts, 45-48 parts and 49-54 parts according to the detection result of thin layer chromatography to obtain a component Fr.M1, a component Fr.M2, a component Fr.M3, a component Fr.M4, a component Fr.M5, a component Fr.M6, a component Fr.M7, a component Fr.M8 and a component Fr.M9;
separating the component Fr.M4 by sephadex LH-20 gel column chromatography, eluting with 5.0L-10L of methanol at a flow rate of 5-10mL/min, collecting 50 fractions each 100-200 mL as one fraction, and respectively combining 1-25 fractions, 26-44 fractions and 45-50 fractions by silica gel thin layer chromatography detection analysis to obtain a subfraction Fr.M4-1, a subfraction Fr.M4-2 and a subfraction Fr.M4-3; the subfraction Fr.M4-1 was separated by ODS column chromatography with MeOH-H in a volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, wherein each gradient is 2.5L-5L, each gradient is a subcomponent, and subcomponent Fr.M4-1-1, subcomponent Fr.M4-1-2, subcomponent Fr.M4-1-3, subcomponent Fr.M4-1-4, subcomponent Fr.M4-1-5 and subcomponent Fr.M4-1-6 are obtained; separating the subcomponent Fr.M4-1-2 by sephadex LH-20 gel column chromatography, eluting with 500-1000mL of methanol at a flow rate of 2.5-5mL/min, collecting 100 fractions each 5mL-10mL as one fraction, respectively combining the fractions 1-17, 18-41, 42-59, 60-77, 78-92 and 93-100 by silica gel thin layer chromatography detection analysis, and collecting to obtain subcomponent Fr.M4-1-2-1, subcomponent Fr.M4-1-2-2, subcomponent Fr.M4-1-2-3, subcomponent Fr.M4-1-2-4 and subcomponent Fr.M4-1-2-4M4-1-2-5, subfraction Fr.M4-1-2-6; purifying the subfraction M4-1-2-3 by preparative high performance liquid chromatography, eluting with se:Sup>A mixed solvent of methanol and water with se:Sup>A volume ratio of 52:48, collecting chromatographic peaks with retention time of 7.9min, 8.3min, 10.8min, 12.8min, 16.4min and 25.1min respectively for the chromatographic column YMC-Pack ODS-A, the flow rate of 3 m/min, and obtaining subfraction Fr.M4-1-2-3-1, subfraction Fr.M4-1-2-3-2, subfraction Fr.M4-1-2-3-3, subfraction Fr.M4-1-2-3-4, subfraction Fr.M4-1-2-3-5 and subfraction Fr.M4-1-2-3-6; purifying the subfraction Fr.M4-1-2-3-3 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peaks with retention time of 17.5min respectively at flow rate of 3 m/min and YMC-Pack ODS-A column to obtain Dysosmse:Sup>A versipellis E (dysosmaflavonoid E);
Purifying the subfraction Fr.M4-1-2-3-4 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peak with flow rate of 3 m/min and retention time of 30.7min to obtain Dysosmse:Sup>A versipellis F (dysosmaflavonoid F);
separating the component Fr.M5 by sephadex LH-20 column chromatography, eluting with 2.5L-5L methanol at a flow rate of 5-10mL/min, collecting 25 fractions each 100mL-200mL as one fraction, and respectively combining 1-3, 4-5, 6-7, 8-10, 11-13, 14-17, 19-21 and 22-25 by silica gel thin layer chromatography detection analysis to obtain a subfraction Fr.M5-1, a subfraction Fr.M5-2, a subfraction Fr.M5-3, a subfraction Fr.M5-4, a subfraction Fr.M5-5, a subfraction Fr.M5-6, a subfraction Fr.M5-7 and a subfraction Fr.M5-8; the subfractions Fr.M5-1 to Fr.M5-5 are combined, separated by MCI column chromatography and separated by MeOH-H with volume ratio of 0:100, 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, each gradient elution solvent is 1.0L-2.0L, the flow rate is 2.5-5.0mL/min, each 250mL-500mL is a fraction, 28 fractions are collected, each fraction is detected and analyzed by silica gel thin layer chromatography, and 1-5 fractions, 6-15 fractions, 16-23 fractions and 17-28 fractions are respectively combined to obtain a subcomponent Fr.M5- 1-1, subfraction Fr.M5-1-2, subfraction Fr.M5-1-3, subfraction Fr.M5-1-4; the subfractions Fr.M5-1-3 were separated by column chromatography on silica gel with CHCl in a volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 7:1, 3:1 3 Gradient elution of a MeOH mixed solvent system, wherein each gradient elution solvent is 50-100 mL, the flow rate is 2.0-5.0mL/min, each 5mL-10mL is one fraction, 80 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-10 fractions, 11-20 fractions, 21-30 fractions, 31-40 fractions, 41-46 fractions, 47-51 fractions, 52-60 fractions, 61-70 fractions and 71-80 fractions are respectively combined to obtain a subfraction Fr.M5-1-3-1, a subfraction Fr.M5-1-3-2, a subfraction Fr.M5-1-3-3, a subfraction Fr.M5-1-3-5, a subfraction Fr.M5-1-3-7, a subfraction Fr.M5-1-3-8, and a subfraction Fr.M5-1-3-9; purifying the subfraction Fr.M5-1-3-5 by preparative high performance liquid chromatography, eluting with methanol-water mixed solvent with volume ratio of 55:45, collecting chromatographic peak with retention time of 34.9min at flow rate of 3 m/min with YMC-Pack ODS-A column to obtain Dysosmse:Sup>A versipellis A (dysosmaflavonoid A);
Separating the component Fr.M6 by sephadex LH-20 column chromatography, eluting with 15L-30L methanol at a flow rate of 5-10mL/min, collecting 30 fractions each 0.5L-1L as one fraction, and respectively combining fractions 1-18 and 19-30 by silica gel thin layer chromatography detection analysis to obtain subfractions Fr.M6-1 and Fr.M6-2; the subfraction Fr.M6-1 is separated by ODS column chromatography with MeOH-H in volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out on the O mixed solvent system, wherein each gradient elution solvent is 2.5L-5L, the flow rate is 3.0-6.0mL/min, each 0.5L-1L is one fraction, 30 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-5 fractions, 6-10 fractions, 11-20 fractions and 21-30 fractions are respectively combined to obtain a subfraction Fr.M6-1-1, a subfraction Fr.M6-1-2, a subfraction Fr.M6-1-3 and a subfraction M6-1-4; the subfractions Fr.M6-1-3 are separated by silica gel column chromatography and mixed solvent of dichloromethane and methanol in volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10 and 100:30Carrying out systematic gradient elution, wherein each gradient elution solvent is 125L-250L, the flow rate is 1.0-2.0mL/min, and each elution gradient is a fraction, so as to obtain a subfraction Fr.M6-1-3-1, a subfraction Fr.M6-1-3-2, a subfraction Fr.M6-1-3-3, a subfraction Fr.M6-1-3-4, a subfraction Fr.M6-1-3-5, a subfraction Fr.M6-1-3-6 and a subfraction Fr.M6-1-3-7; separating the subfractions Fr.M6-1-3-4 by sephadex LH-20 gel column chromatography, eluting with 125mL-250mL of methanol at a flow rate of 1-2mL/min, collecting 25 fractions each of which is 5mL-10mL, and respectively combining the fractions 1-7, 8-16 and 17-25 by silica gel thin layer chromatography detection analysis to obtain subfractions Fr.M6-1-3-4-1, subfractions Fr.M6-1-3-4-2 and subfractions Fr.M6-1-3-4-3; purifying the subfraction Fr.M6-1-3-4-2 by preparative high performance liquid chromatography, eluting with methanol-water mixed solvent with volume ratio of 50:50, collecting chromatographic peaks with retention time of 33.1min, 41.5min and 45.0min respectively for the chromatographic column YMC-Pack ODS-A and flow rate of 3 m/min, and obtaining subfraction Fr.M6-1-3-4-2-1, subfraction Fr.M6-1-3-4-2-2 and subfraction Fr.M6-1-3-4-2-3; purifying the subfraction Fr.M6-1-3-4-2-1 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 35:65, collecting chromatographic peak with flow rate of 3 m/min and retention time of 11.9min to obtain compound dysosmse:Sup>A versipellis C (dysosmaflavonoid C);
Purifying the subfraction Fr.M6-1-3-4-2-2 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 30:70, collecting chromatographic peak with flow rate of 3 m/min and retention time of 15.6min to obtain compound dysosmse:Sup>A versipellis D (dysosmaflavonoid D);
purifying the subfraction Fr.M6-1-3-4-2-3 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 30:70, collecting chromatographic peak with flow rate of 3 m/min and retention time of 28.5min, and collecting the compound dysosmse:Sup>A versipellis B (dysosmaflavonoid B).
The novel flavonol compounds dysosma versipellis A-F with antioxidant activity, which are prepared by the invention, can be used for preparing antioxidants for foods or beverages, and has the advantages of abundant raw materials, easy operation of the preparation method and remarkable economic and social benefits.
Drawings
FIG. 1 is a diagram of the molecular structure of the present invention.
FIG. 2 shows dysosma versipellis A of the present invention 1 H NMR spectrum (500 MHz).
FIG. 3 shows dysosma versipellis A of the present invention 13 C NMR spectrum (125 MHz).
FIG. 4 is a 1H NMR spectrum (500 MHz) of dysosma versipellis B of the invention.
FIG. 5 shows dysosma versipellis B of the present invention 13 C NMR spectrum (125 MHz).
FIG. 6 shows dysosma versipellis C of the present invention 1 H NMR spectrum (500 MHz).
FIG. 7 shows dysosma versipellis C of the present invention 13 C NMR spectrum (125 MHz).
FIG. 8 shows dysosma versipellis D of the present invention 1 H NMR spectrum (500 MHz).
FIG. 9 shows dysosma versipellis D of the present invention 13 C NMR spectrum (125 MHz).
FIG. 10 shows dysosma versipellis E of the present invention 1 H NMR spectrum (500 MHz).
FIG. 11 shows dysosma versipellis E of the present invention 13 C NMR spectrum (125 MHz).
FIG. 12 shows dysosma versipellis F of the present invention 1 H NMR spectrum (500 MHz).
FIG. 13 shows dysosma versipellis F of the present invention 13 C NMR spectrum (125 MHz).
Detailed Description
The following describes specific embodiments of the present invention in detail with reference to examples.
The invention may be embodied by the following examples.
Example 1
The invention relates to a preparation method of flavonol compound dysosma versipellis ketone A-F, which takes 40kg of dysosma versipellis as raw material, adds 3 times of ethanol with weight volume and volume concentration of 95% of the raw material each time, and carries out heating reflux extraction for 3 times, wherein the extraction temperature is 95 ℃ and the extraction time each timeRecovering 95% ethanol under reduced pressure for 1 hr to obtain extract 95% ethanol extract; adding 3 times of ethanol with weight volume and volume concentration of 50% into the residue, reflux extracting for 1 time at 95deg.C for 1 hr, and recovering 50% ethanol under reduced pressure to obtain extract of 50% ethanol; combining 95% ethanol extract and 50% ethanol extract (5.4 kg), adding 10.8L absolute ethanol for dissolution, adding 10.8kg diatomite for adsorption, recovering solvent, sequentially adding 16.2L dichloromethane, ethyl acetate and methanol for elution, and recovering solvent to obtain dichloromethane elution part, ethyl acetate elution part and methanol elution part respectively; separating methanol elution part (3.4 kg) by silica gel column chromatography, sequentially performing gradient elution by using a dichloromethane-methanol mixed solvent system with volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:30, 100:50 and 0:100, wherein each gradient is prepared by using 60L eluent with flow rate of 70mL/min, each 10L is a fraction, collecting 54 fractions, detecting and analyzing each fraction by silica gel thin layer chromatography, and using GF 254 The thin layer plate is heated for 4min at 105 ℃ by taking petroleum ether-acetone with the volume ratio of 4:5 and dichloromethane-methanol with the volume ratio of 5:2 as developing agents, respectively, and according to the detection result of thin layer chromatography, 1-12 parts, 13-24 parts, 25-30 parts, 31-36 parts, 37-38 parts, 39-42 parts, 43-44 parts, 45-48 parts and 49-54 parts are combined respectively to obtain a component Fr.M1, a component Fr.M2, a component Fr.M3, a component Fr.M4, a component Fr.M5, a component Fr.M6, a component Fr.M7, a component Fr.M8 and a component Fr.M9;
separating the component Fr.M4 (90.2 g) by sephadex LH-20 gel column chromatography, eluting with 10L of methanol at a flow rate of 10mL/min, collecting 50 fractions each 200mL as one fraction, and respectively combining fractions 1-25, 26-44 and 45-50 by silica gel thin layer chromatography detection analysis to obtain a subfraction Fr.M4-1, a subfraction Fr.M4-2 and a subfraction Fr.M4-3; the subfraction Fr.M4-1 (23.7 g) was separated by ODS column chromatography with MeOH-H in a volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, each gradient is 5L, each gradient is a subcomponent, and subcomponent Fr.M4- 1-1, subfraction Fr.M4-1-2, subfraction Fr.M4-1-3, subfraction Fr.M4-1-4, subfraction Fr.M4-1-5, subfraction Fr.M4-1-6; separating the subfractions Fr.M4-1-2 (4.8 g) by sephadex LH-20 gel column chromatography, eluting with 1000mL of methanol, wherein the flow rate is 5mL/min, each 10mL is one fraction, collecting 100 fractions, respectively combining the fractions 1-17, 18-41, 42-59, 60-77, 78-92 and 93-100 by silica gel thin layer chromatography detection analysis, and collecting the fractions to obtain subfractions Fr.M4-1-2-1, subfractions Fr.M4-1-2-2, subfractions Fr.M4-1-2-3, subfractions Fr.M4-1-2-4, subfractions Fr.M4-1-2-5 and subfractions Fr.M4-1-2-6; purifying the subfraction M4-1-2-3 (1.6 g) by preparative high performance liquid chromatography, eluting with se:Sup>A mixed methanol-water solvent with se:Sup>A volume ratio of 52:48, collecting chromatographic peaks with retention time of 7.9min, 8.3min, 10.8min, 12.8min, 16.4min and 25.1min respectively, and obtaining subfraction Fr.M4-1-2-3-1, subfraction Fr.M4-1-2-3-2, subfraction Fr.M4-1-2-3-3, subfraction Fr.M4-1-2-3-4, subfraction Fr.M4-1-2-3-5 and subfraction Fr.M4-1-2-3-6; purifying subfraction Fr.M4-1-2-3-3 (20.5 mg) by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peaks with retention time of 17.5min at flow rate of 3 m/min with YMC-Pack ODS-A chromatography column to obtain dysosmse:Sup>A versipellis E (dysosmaflavonoid E,3.0 mg);
Purifying subfraction Fr.M4-1-2-3-4 (17.5 mg) by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peak with retention time of 30.7min at flow rate of 3 m/min with YMC-Pack ODS-A to obtain dysosmse:Sup>A versipellis ketone F (dysosmaflavonoid F,4.0 mg);
separating component Fr.M5 (110.0 g) by sephadex LH-20 column chromatography, eluting with 5L methanol at a flow rate of 10mL/min, collecting 25 fractions per 200mL, and subjecting each fraction to silica gel thin layer chromatography detection analysis to obtain subgroups 1-3, 4-5, 6-7, 8-10, 11-13, 14-17, 19-21, 22-25The composition comprises the following components of part Fr.M5-1, sub-component Fr.M5-2, sub-component Fr.M5-3, sub-component Fr.M5-4, sub-component Fr.M5-5, sub-component Fr.M5-6, sub-component Fr.M5-7 and sub-component Fr.M5-8; the subfractions Fr.M5-1 to Fr.M5-5 (22.9 g) were combined, separated by MCI column chromatography, and purified by MeOH-H in a volume ratio of 0:100, 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Carrying out gradient elution on an O mixed solvent system, wherein each gradient elution solvent is 2.0L, the flow rate is 5.0mL/min, each 500mL is a fraction, 28 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-5, 6-15, 16-23 and 17-28 of the fractions are respectively combined to obtain a subfraction Fr.M5-1, a subfraction Fr.M5-1-2, a subfraction Fr.M5-1-3 and a subfraction Fr.M5-1-4; the subfractions Fr.M5-1-3 (2.5 g) were separated by column chromatography on silica gel with a volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 7:1, 3:1 CHCl 3 Gradient elution is carried out by a MeOH mixed solvent system, wherein each gradient elution solvent is 100mL, the flow rate is 5.0mL/min, each 10mL is one fraction, 80 fractions are collected, each fraction is subjected to silica gel thin layer chromatography detection analysis, and 1-10 fractions, 11-20 fractions, 21-30 fractions, 31-40 fractions, 41-46 fractions, 47-51 fractions, 52-60 fractions, 61-70 fractions and 71-80 fractions are respectively combined to obtain subfractions Fr.M5-1-3-1, subfractions Fr.M5-1-3-2, subfractions Fr.M5-1-3-3, subfractions Fr.M5-1-4, subfractions Fr.M5-1-3-5, subfractions Fr.M5-1-3-6, subfractions Fr.M5-1-3-8, subfractions Fr.M5-1-3-9; purifying subfraction Fr.M5-1-3-5 (0.25 g) by preparative high performance liquid chromatography, eluting with methanol-water mixed solvent with volume ratio of 55:45, collecting chromatographic peak with retention time of 34.9min at flow rate of 3 m/min with YMC-Pack ODS-A to obtain dysosmse:Sup>A versipellis A (dysosmaflavonoid A,5.0 mg);
separating the component Fr.M6 (130.0 g) by sephadex LH-20 column chromatography, eluting with 30L of methanol at a flow rate of 10mL/min, collecting 30 fractions each 1L as one fraction, and respectively combining fractions 1-18 and 19-30 by silica gel thin layer chromatography detection analysis to obtain subfractions Fr.M6-1 and Fr.M6-2; the subfraction Fr.M6-1 (57.3 g) was separated by ODS column chromatography to give the body MeOH-H with product ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, wherein each gradient elution solvent is 5L, the flow rate is 6.0mL/min, each 1L is one fraction, 30 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-5 fractions, 6-10 fractions, 11-20 fractions and 21-30 fractions are respectively combined to obtain a subfraction Fr.M6-1-1, a subfraction Fr.M6-1-2, a subfraction Fr.M6-1-3 and a subfraction M6-1-4; separating the subfractions Fr.M6-1-3 (5.9 g) by silica gel column chromatography, and performing gradient elution by using a methylene dichloride-methanol mixed solvent system with the volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10 and 100:30, wherein each gradient elution solvent is 250L, the flow rate is 2.0mL/min, and each elution gradient is one flow, so as to obtain subfractions Fr.M6-1-3-1, subfractions Fr.M6-1-3-2, subfractions Fr.M6-1-3-3, subfractions Fr.M6-1-3-4, subfractions Fr.M6-1-3-5, subfractions Fr.M6-1-3-6 and subfractions Fr.M6-1-3-7; separating subfractions Fr.M6-1-3-4 (3.2 g) by sephadex LH-20 gel column chromatography, eluting with 250mL of methanol, wherein the flow rate is 2mL/min, each 10mL is a fraction, collecting 25 fractions, and respectively combining fractions 1-7, 8-16 and 17-25 by silica gel thin layer chromatography detection analysis to obtain subfractions Fr.M6-1-3-4-1, subfractions Fr.M6-1-3-4-2 and subfractions Fr.M6-1-3-4-3; purifying the subfraction Fr.M6-1-3-4-2 (0.99 g) by preparative high performance liquid chromatography, eluting with se:Sup>A methanol-water mixed solvent with se:Sup>A volume ratio of 50:50, collecting chromatographic peaks with retention time of 33.1min, 41.5min and 45.0min respectively at se:Sup>A flow rate of 3 m/min for YMC-Pack ODS-A, and collecting subfraction Fr.M6-1-3-4-2-1, subfraction Fr.M6-1-3-4-2-2 and subfraction Fr.M6-1-3-4-2-3; purifying subfraction Fr.M6-1-3-4-2-1 (20.6 mg) by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 35:65, collecting chromatographic peak with flow rate of 3 m/min and retention time of 11.9min to obtain compound dysosmse:Sup>A versipellis C (dysosmaflavonoid C,7.0 mg);
Purifying subfraction Fr.M6-1-3-4-2-2 (7.6 mg) by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 30:70, collecting chromatographic peak with flow rate of 3 m/min and retention time of 15.6min to obtain compound dysosmse:Sup>A versipellis D (dysosmaflavonoid D,3.9 mg);
the subfraction Fr.M6-1-3-4-2-3 (16.5 mg) is purified by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 30:70, and collecting chromatographic peak with retention time of 28.5min at flow rate of 3 m/min with YMC-Pack ODS-A to obtain compound dysosmse:Sup>A versipellis B (dysosmaflavonoid B,7.9 mg).
Example 2
The invention relates to a preparation method of flavonol compound dysosma versipellis ketone A-F, which takes 20kg of dysosma versipellis medicinal material as raw material, adds 5 times of ethanol with the weight volume and the volume concentration of 95% of the raw material each time, carries out heating reflux extraction for 3 times, the extraction temperature is 92 ℃, the extraction time is 1.5 hours each time, and decompresses and recovers 95% of ethanol to obtain extract of 95% of ethanol; adding ethanol with weight volume of 5 times and volume concentration of 50% into the residue, reflux extracting for 1 time at 92 deg.C for 1.5 hr, and recovering 50% ethanol under reduced pressure to obtain extract 50% ethanol extract; combining 95% ethanol extract and 50% ethanol extract (2.7 kg), adding 5.4L absolute ethanol for dissolution, adding 5.4kg diatomite for adsorption, recovering solvent, sequentially adding 8.1L dichloromethane, ethyl acetate and methanol for elution, and recovering solvent to obtain dichloromethane elution part, ethyl acetate elution part and methanol elution part respectively; separating methanol elution part (1.7 kg) by silica gel column chromatography, sequentially performing gradient elution by using a dichloromethane-methanol mixed solvent system with volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:30, 100:50 and 0:100, wherein each gradient is prepared by using 30L eluent with flow rate of 50mL/min, each 5L is a fraction, collecting 54 fractions, detecting and analyzing each fraction by silica gel thin layer chromatography, and performing GF detection and analysis on each fraction 254 The thin layer plate is prepared by respectively taking petroleum ether-acetone with the volume ratio of 4:5 and dichloromethane-methanol with the volume ratio of 5:2 as developing agents, taking sulfuric acid-ethanol solution with the volume ratio of 10:90 as a color developing agent, heating at 105 ℃ for 5min, respectively combining 1-12 parts by volume, 13-24 parts by volume, 25-30 parts by volume, and,31-36 parts, 37-38 parts, 39-42 parts, 43-44 parts, 45-48 parts and 49-54 parts to obtain a component Fr.M1, a component Fr.M2, a component Fr.M3, a component Fr.M4, a component Fr.M5, a component Fr.M6, a component Fr.M7, a component Fr.M8 and a component Fr.M9;
separating the component Fr.M4 (45.0 g) by sephadex LH-20 gel column chromatography, eluting with 5.0L methanol at a flow rate of 5mL/min, collecting 50 fractions each 100mL as one fraction, and respectively combining fractions 1-25, 26-44 and 45-50 by silica gel thin layer chromatography detection analysis to obtain subfractions Fr.M4-1, subfractions Fr.M4-2 and subfractions Fr.M4-3; the subfraction Fr.M4-1 (11.8 g) was separated by ODS column chromatography with MeOH-H in a volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, wherein each gradient is 2.5L, each gradient is a subcomponent, and subcomponent Fr.M4-1-1, subcomponent Fr.M4-1-2, subcomponent Fr.M4-1-3, subcomponent Fr.M4-1-4, subcomponent Fr.M4-1-5 and subcomponent Fr.M4-1-6 are obtained; separating the subfractions Fr.M4-1-2 (2.4 g) by sephadex LH-20 gel column chromatography, eluting with 500mL of methanol, wherein the flow rate is 2.5mL/min, each 5mL is a fraction, collecting 100 fractions, respectively combining fractions 1-17, 18-41, 42-59, 60-77, 78-92 and 93-100 by silica gel thin layer chromatography detection analysis, and collecting subfractions Fr.M4-1-2-1, subfractions Fr.M4-1-2-2, subfractions Fr.M4-1-2-3, subfractions Fr.M4-1-2-4, subfractions Fr.M4-1-2-5 and subfractions Fr.M4-1-2-6; purifying the subfraction M4-1-2-3 (0.8 g) by preparative high performance liquid chromatography, eluting with se:Sup>A mixed methanol-water solvent with se:Sup>A volume ratio of 52:48, collecting chromatographic peaks with retention time of 7.9min, 8.3min, 10.8min, 12.8min, 16.4min and 25.1min respectively, and obtaining subfraction Fr.M4-1-2-3-1, subfraction Fr.M4-1-2-3-2, subfraction Fr.M4-1-2-3-3, subfraction Fr.M4-1-2-3-4, subfraction Fr.M4-1-2-3-5 and subfraction Fr.M4-1-2-3-6; the subfraction Fr.M4-1-2-3-3 is purified by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with the volume ratio of 27:73, The chromatographic column is YMC-Pack ODS-A, the flow rate is 3 m/min, and chromatographic peaks with retention time of 17.5min are respectively collected to obtain dysosmse:Sup>A versipellis E (dysosmaflavonoid E,1.5 mg);
purifying subfraction Fr.M4-1-2-3-4 (8.7 mg) by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peak with retention time of 30.7min at flow rate of 3 m/min with YMC-Pack ODS-A column to obtain dysosmse:Sup>A versipellis ketone F (dysosmaflavonoid F,2.0 mg);
separating the component Fr.M5 (55.0 g) by sephadex LH-20 column chromatography, eluting with 2.5L of methanol, wherein the flow rate is 5mL/min, taking 100mL as one fraction, collecting 25 fractions, respectively combining 1-3 fractions, 4-5 fractions, 6-7 fractions, 8-10 fractions, 11-13 fractions, 14-17 fractions, 19-21 fractions and 22-25 fractions by silica gel thin layer chromatography detection analysis to obtain a subfraction Fr.M5-1, a subfraction Fr.M5-2, a subfraction Fr.M5-3, a subfraction Fr.M5-4, a subfraction Fr.M5-5, a subfraction Fr.M5-6, a subfraction Fr.M5-7 and a subfraction Fr.M5-8; the subfractions Fr.M5-1 to Fr.M5-5 (11.4 g) were combined, separated by MCI column chromatography, and purified by MeOH-H in a volume ratio of 0:100, 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Carrying out gradient elution on an O mixed solvent system, wherein each gradient elution solvent is 1.0L, the flow rate is 2.5mL/min, each 250mL is one fraction, 28 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-5, 6-15, 16-23 and 17-28 of the fractions are respectively combined to obtain a subfraction Fr.M5-1, a subfraction Fr.M5-1-2, a subfraction Fr.M5-1-3 and a subfraction Fr.M5-1-4; the subfractions Fr.M5-1-3 (1.2 g) were separated by column chromatography on silica gel with a volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 7:1, 3:1 CHCl 3 Gradient elution of MeOH mixed solvent systems, wherein each gradient elution solvent is 50mL, the flow rate is 2.0mL/min, each 5mL is one fraction, 80 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-10, 11-20, 21-30, 31-40, 41-46, 47-51, 52-60, 61-70 and 71-80 of the fractions are respectively combined to obtain a subfraction Fr.M5-1-3-1, a subfraction Fr.M5-1-3-2 and a subfraction Fr.M5-1-3-2Fr.m5-1-3-3, subfraction fr.m5-1-3-4, subfraction fr.m5-1-3-5, subfraction fr.m5-1-3-6, subfraction fr.m5-1-3-7, subfraction fr.m5-1-3-8, subfraction fr.m5-1-3-9; purifying subfraction Fr.M5-1-3-5 (0.13 g) by preparative high performance liquid chromatography, eluting with methanol-water mixed solvent with volume ratio of 55:45, collecting chromatographic peak with retention time of 34.9min at flow rate of 3 m/min with YMC-Pack ODS-A to obtain dysosmse:Sup>A versipellis A (dysosmaflavonoid A,2.5 mg);
Separating the component Fr.M6 (65.0 g) by sephadex LH-20 column chromatography, eluting with 15L methanol at a flow rate of 5mL/min, collecting 30 fractions each 0.5L as one fraction, and respectively combining fractions 1-18 and 19-30 by silica gel thin layer chromatography detection analysis to obtain sub-components Fr.M6-1 and Fr.M6-2; the subfraction Fr.M6-1 (28.6 g) was separated by ODS column chromatography with MeOH-H in a volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, each gradient elution solvent is 2.5L, the flow rate is 3.0mL/min, each 0.5L is one fraction, 30 fractions are collected, each fraction is detected and analyzed by silica gel thin layer chromatography, and 1-5 fractions, 6-10 fractions, 11-20 fractions and 21-30 fractions are combined to obtain a subfraction Fr.M6-1-1, a subfraction Fr.M6-1-2, a subfraction Fr.M6-1-3 and a subfraction M6-1-4; separating the subfractions Fr.M6-1-3 (2.9 g) by silica gel column chromatography, and performing gradient elution by using a methylene dichloride-methanol mixed solvent system with the volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10 and 100:30, wherein each gradient elution solvent is 125L, the flow rate is 1.0mL/min, and each elution gradient is one flow, so as to obtain subfractions Fr.M6-1-3-1, subfractions Fr.M6-1-3-2, subfractions Fr.M6-1-3-3, subfractions Fr.M6-1-3-4, subfractions Fr.M6-1-3-5, subfractions Fr.M6-1-3-6 and subfractions Fr.M6-1-3-7; separating subcomponent Fr.M6-1-3-4 (1.6 g) by sephadex LH-20 gel column chromatography, eluting with 125mL of methanol at a flow rate of 1mL/min, collecting 25 fractions each of which is one fraction per 5mL, and subjecting each fraction to silica gel thin layer chromatography detection analysis to obtain subcomponent Fr.M6-1-3-4-1, subcomponent Fr.M6-1-3-4-2, Subfraction Fr.M6-1-3-4-3; purifying the subfraction Fr.M6-1-3-4-2 (0.5 g) by preparative high performance liquid chromatography, eluting with se:Sup>A methanol-water mixed solvent with se:Sup>A volume ratio of 50:50, collecting chromatographic peaks with retention time of 33.1min, 41.5min and 45.0min respectively at se:Sup>A flow rate of 3 m/min for YMC-Pack ODS-A, and obtaining subfraction Fr.M6-1-3-4-2-1, subfraction Fr.M6-1-3-4-2-2 and subfraction Fr.M6-1-3-4-2-3; purifying subfraction Fr.M6-1-3-4-2-1 (10.3 mg) by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 35:65, collecting chromatographic peak with flow rate of 3 m/min and retention time of 11.9min to obtain compound dysosmse:Sup>A versipellis C (dysosmaflavonoid C,3.5 mg);
purifying subfraction Fr.M6-1-3-4-2-2 (3.8 mg) by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 30:70, collecting chromatographic peak with flow rate of 3 m/min and retention time of 15.6min to obtain compound dysosmse:Sup>A versipellis D (dysosmaflavonoid D,2.0 mg);
the subfraction Fr.M6-1-3-4-2-3 (8.2 mg) is purified by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 30:70, and collecting chromatographic peak with retention time of 28.5min at flow rate of 3 m/min with YMC-Pack ODS-A to obtain compound dysosmse:Sup>A versipellis B (dysosmaflavonoid B,4.0 mg).
Example 3
The invention relates to a preparation method of flavonol compound dysosma versipellis ketone A-F, which takes 30kg of dysosma versipellis medicinal material as raw material, adds 4 times of ethanol with the weight volume and the volume concentration of 95% of the raw material each time, carries out heating reflux extraction for 3 times, the extraction temperature is 93 ℃, the extraction time is 1.2 hours each time, and decompresses and recovers 95% ethanol to obtain extract of 95% ethanol; adding 4 times of ethanol with weight volume and volume concentration of 50% into the residue, reflux extracting for 1 time at 93 deg.C for 1.2 hr, and recovering 50% ethanol under reduced pressure to obtain extract 50% ethanol extract; combining 95% ethanol extract and 50% ethanol extract (4.0 kg), dissolving in 8.0L anhydrous ethanol, adsorbing with 8.0kg diatomite, and recoveringAfter the solvent is recovered, 12L of dichloromethane, ethyl acetate and methanol are sequentially added for eluting, and the solvent is recovered to obtain a dichloromethane eluting part, an ethyl acetate eluting part and a methanol eluting part respectively; separating methanol elution part (2.5 kg) by silica gel column chromatography, sequentially performing gradient elution by using a dichloromethane-methanol mixed solvent system with volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:30, 100:50 and 0:100, wherein each gradient is prepared by using 45L eluent with flow rate of 60mL/min and each 7.5L is prepared as a fraction, collecting 54 fractions, detecting and analyzing each fraction by silica gel thin layer chromatography, and performing GF detection analysis on each fraction 254 The thin layer plate is heated for 3min at 105 ℃ by taking petroleum ether-acetone with the volume ratio of 4:5 and dichloromethane-methanol with the volume ratio of 5:2 as developing agents, respectively, and according to the detection result of thin layer chromatography, 1-12 parts, 13-24 parts, 25-30 parts, 31-36 parts, 37-38 parts, 39-42 parts, 43-44 parts, 45-48 parts and 49-54 parts are combined to obtain a component Fr.M1, a component Fr.M2, a component Fr.M3, a component Fr.M4, a component Fr.M5, a component Fr.M6, a component Fr.M7, a component Fr.M8 and a component Fr.M9;
separating the component Fr.M4 (67.6 g) by sephadex LH-20 gel column chromatography, eluting with 7.5L methanol at a flow rate of 7.5mL/min, collecting 50 fractions each 150mL as one fraction, and respectively combining fractions 1-25, 26-44 and 45-50 by silica gel thin layer chromatography detection analysis to obtain a subfraction Fr.M4-1, a subfraction Fr.M4-2 and a subfraction Fr.M4-3; the subfraction Fr.M4-1 (17.7 g) was separated by ODS column chromatography with MeOH-H in a volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out on the O mixed solvent system, each gradient is 3.75L, each gradient is a subcomponent, and subcomponent Fr.M4-1-1, subcomponent Fr.M4-1-2, subcomponent Fr.M4-1-3, subcomponent Fr.M4-1-4, subcomponent Fr.M4-1-5 and subcomponent Fr.M4-1-6 are obtained; separating subfraction Fr.M4-1-2 (3.6 g) by sephadex LH-20 gel column chromatography, eluting with 750mL methanol at a flow rate of 4mL/min, collecting 100 fractions each of which is a fraction of 7.5mL, and combining fractions 1-17, 18-41, 42-59, and fraction respectively by silica gel thin layer chromatography detection analysis 60-77, 78-92 and 93-100, collecting the obtained subfractions Fr.M4-1-2-1, fr.M4-1-2-2, fr.M4-1-2-3, fr.M4-1-2-4, fr.M4-1-2-5 and Fr.M4-1-2-6; purifying the subfraction M4-1-2-3 (1.2 g) by preparative high performance liquid chromatography, eluting with se:Sup>A mixed methanol-water solvent with se:Sup>A volume ratio of 52:48, collecting chromatographic peaks with retention time of 7.9min, 8.3min, 10.8min, 12.8min, 16.4min and 25.1min respectively, and obtaining subfraction Fr.M4-1-2-3-1, subfraction Fr.M4-1-2-3-2, subfraction Fr.M4-1-2-3-3, subfraction Fr.M4-1-2-3-4, subfraction Fr.M4-1-2-3-5 and subfraction Fr.M4-1-2-3-6; purifying subfraction Fr.M4-1-2-3-3 (15.3 mg) by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peaks with retention time of 17.5min at flow rate of 3 m/min with YMC-Pack ODS-A chromatography column to obtain dysosmse:Sup>A versipellis E (dysosmaflavonoid E,1.6 mg);
purifying subfraction Fr.M4-1-2-3-4 (13.2 mg) by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peak with retention time of 30.7min at flow rate of 3 m/min with YMC-Pack ODS-A to obtain dysosmse:Sup>A versipellis ketone F (dysosmaflavonoid F,2.2 mg);
Separating the component Fr.M5 (82.5 g) by sephadex LH-20 column chromatography, eluting with 3.75L of methanol, wherein the flow rate is 7.5mL/min, each 150mL is one fraction, collecting 25 fractions, and respectively combining 1-3 fractions, 4-5 fractions, 6-7 fractions, 8-10 fractions, 11-13 fractions, 14-17 fractions, 19-21 fractions and 22-25 fractions by silica gel thin layer chromatography detection analysis to obtain a subfraction Fr.M5-1, a subfraction Fr.M5-2, a subfraction Fr.M5-3, a subfraction Fr.M5-4, a subfraction Fr.M5-5, a subfraction Fr.M5-6, a subfraction Fr.M5-7 and a subfraction Fr.M5-8; the subfractions Fr.M5-1 to Fr.M5-5 (17.1 g) were combined, separated by MCI column chromatography, and purified by MeOH-H in a volume ratio of 0:100, 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, wherein each gradient elution solvent is 1.5L, and the flow rate is 13.7mL/min, wherein each 375mL is a fraction, 28 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-5, 6-15, 16-23 and 17-28 are respectively combined to obtain a subfraction Fr.M5-1-1, a subfraction Fr.M5-1-2, a subfraction Fr.M5-1-3 and a subfraction Fr.M5-1-4; the subfractions Fr.M5-1-3 (1.8 g) were separated by column chromatography on silica gel with a volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 7:1, 3:1 CHCl 3 Gradient elution of MeOH mixed solvent systems, wherein each gradient elution solvent is 75mL, the flow rate is 4.0mL/min, each 7.5mL is one fraction, 80 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-10 fractions, 11-20 fractions, 21-30 fractions, 31-40 fractions, 41-46 fractions, 47-51 fractions, 52-60 fractions, 61-70 fractions and 71-80 fractions are respectively combined to obtain subfractions Fr.M5-1-3-1, subfractions Fr.M5-1-3-2, subfractions Fr.M5-1-3-3, subfractions Fr.M5-1-3-4, subfractions Fr.M5-1-3-5, subfractions Fr.M5-1-3-6, subfractions Fr.M5-1-3-7, subfractions Fr.M5-1-3-8 and subfractions Fr.M5-1-3-9; purifying subfraction Fr.M5-1-3-5 (0.18 g) by preparative high performance liquid chromatography, eluting with methanol-water mixed solvent with volume ratio of 55:45, collecting chromatographic peak with retention time of 34.9min at flow rate of 3 m/min with YMC-Pack ODS-A to obtain dysosmse:Sup>A versipellis A (dysosmaflavonoid A,3.7 mg);
separating the component Fr.M6 (97.5 g) by sephadex LH-20 column chromatography, eluting with 22.5L methanol at a flow rate of 7.5mL/min, collecting 30 fractions each 0.75L as one fraction, and respectively combining fractions 1-18 and 19-30 by silica gel thin layer chromatography detection analysis to obtain subfractions Fr.M6-1 and Fr.M6-2; the subfraction Fr.M6-1 (42.9 g) was separated by ODS column chromatography with MeOH-H in a volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, each gradient elution solvent is 3.75L, the flow rate is 5.0mL/min, each 0.75L is one fraction, 30 fractions are collected, each fraction is detected and analyzed by silica gel thin layer chromatography, and 1-5 fractions, 6-10 fractions, 11-20 fractions and 21-30 fractions are respectively combined to obtain a subfraction Fr.M6-1-1, a subfraction Fr.M6-1-2 and a subfractionA fraction Fr.M6-1-3, a subfraction M6-1-4; separating the subfractions Fr.M6-1-3 (4.4 g) by silica gel column chromatography, and performing gradient elution by using a methylene dichloride-methanol mixed solvent system with the volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10 and 100:30, wherein each gradient elution solvent is 185L, the flow rate is 1.5mL/min, and each elution gradient is one flow, so as to obtain subfractions Fr.M6-1-3-1, subfractions Fr.M6-1-3-2, subfractions Fr.M6-1-3-3, subfractions Fr.M6-1-3-4, subfractions Fr.M6-1-3-5, subfractions Fr.M6-1-3-6 and subfractions Fr.M6-1-3-7; separating subcomponent Fr.M6-1-3-4 (2.4 g) by sephadex LH-20 gel column chromatography, eluting with 175mL of methanol at a flow rate of 1.5mL/min, collecting 25 fractions each 7mL of which is a fraction, and respectively combining fraction 1-7, fraction 8-16 and fraction 17-25 by silica gel thin layer chromatography detection analysis to obtain subcomponent Fr.M6-1-3-4-1, subcomponent Fr.M6-1-3-4-2 and subcomponent Fr.M6-1-3-4-3; purifying the subfraction Fr.M6-1-3-4-2 (0.74 g) by preparative high performance liquid chromatography, eluting with se:Sup>A methanol-water mixed solvent with se:Sup>A volume ratio of 50:50, collecting chromatographic peaks with retention time of 33.1min, 41.5min and 45.0min respectively at se:Sup>A flow rate of 3 m/min for YMC-Pack ODS-A, and collecting subfraction Fr.M6-1-3-4-2-1, subfraction Fr.M6-1-3-4-2-2 and subfraction Fr.M6-1-3-4-2-3; purifying subfraction Fr.M6-1-3-4-2-1 (15.4 mg) by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 35:65, collecting chromatographic peak with flow rate of 3 m/min and retention time of 11.9min to obtain compound dysosmse:Sup>A versipellis C (dysosmaflavonoid C,5.2 mg);
Purifying subfraction Fr.M6-1-3-4-2-2 (5.7 mg) by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 30:70, collecting chromatographic peak with flow rate of 3 m/min and retention time of 15.6min to obtain compound dysosmse:Sup>A versipellis D (dysosmaflavonoid D,2.9 mg);
the subfraction Fr.M6-1-3-4-2-3 (12.4 mg) is purified by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 30:70, and collecting chromatographic peak with retention time of 28.5min at flow rate of 3 m/min with YMC-Pack ODS-A to obtain compound dysosmse:Sup>A versipellis B (dysosmaflavonoid B,5.9 mg).
The compounds prepared by the methods in examples 1-3 of the present invention were subjected to IR, UV and nuclear magnetic resonance spectroscopy 1 H-NMR、 13 C-NMR, HSQC, HMBC), high resolution mass spectrometry (HR-ESI-MS) and the like, are identified as dysosma versipellis A (dysosmaflavonoid A), dysosma versipellis B (dysosmaflavonoid B), dysosma versipellis C (dysosmaflavonoid C), dysosma versipellis D (dysosmaflavonoid D), dysosma versipellis E (dysosmaflavonoid E), dysosma versipellis F (dysosmaflavonoid F), respectively. The related maps are shown in figures 2-13. Experiments show that the method is stable and reliable and is easy to operate, and the obtained product has the capability of scavenging DPPH free radicals. The related data are as follows:
1. Structural identification of compounds
Compound 1 was a yellow amorphous powder. HR-ESI-MS gives an excimer ion peak M/z: [ M+H ]] + 477.1028 (calculated as 477.1033), molecular formula C was determined 22 H 21 O 12 The unsaturation was 13. Infrared spectra showed the presence of two carbonyl groups (1743, 1653 cm) -1 ) Hydroxyl (3380 cm) -1 ) Benzene ring (1597, 1499 cm) -1 ). The ultraviolet spectrum gives a characteristic maximum absorption of flavonols of 244nm, 363nm. 1 H NMR(500MHz,DMSO-d 6 ) In delta H 8.07 (2 h, d, j=8.8 Hz), 6.95 (2 h, d, j=8.8 Hz) is AA ' BB ' system, characteristic hydrogen signal of flavonol B ring, suggesting 4' -oxygen substitution on B ring. Delta 6.84 (1 h, d, j=1.9 Hz) and delta 6.45 (1 h, d, j=2.0 Hz) are meta-coupled, i.e. are characteristic hydrogen signals of the flavonol a ring, and the chemical shift increases, suggesting 5, 7-dioxygen substitution on the a ring. Delta 5.33 (1 h, d, j=7.3 Hz) is a glucose end group hydrogen proton signal, the relative configuration being determined to be beta based on its coupling constant. The absolute configuration of glucose is shown to be D-type by acid hydrolysis and HPLC analysis. Delta 3.67 (3H, s) is a methoxy signal. 13 C NMR(125MHz，DMSO-d 6 ) A total of 22 carbon signals are given, 13 c NMR and HSQC spectra showed a set of beta-glucuronic acid carbon signals delta C 99.1, 72.8, 75.5, 71.4, 75.2, 169.3; methoxy carbon letterNumber delta C 52.0; a flavonol skeleton, wherein delta C 176.1 is a characteristic signal of the carbonyl group at the 4-position of flavonol. The 5-hydrogen proton signal δ4.22 (1 h, d, j=9.5 Hz) and the methoxy hydrogen signal δ3.67 (3 h, s) of β -glucose are both identical to the carbonyl carbon signal δ C HMBC remote correlation of 169.3 indicates methyl esterification of the 6-position of beta-glucuronic acid. The hydrogen signal delta 5.32 (1H, d, J=7.3 Hz) and the flavonol mother nucleus carbon signal delta are terminated by glucuronic acid ester C 162.2 The HMBC remote correlation of (C-7) indicates that the β -glucuronate group is attached at the 7-position. According to 1 H-NMR 13 C-NMR, data were specifically assigned in combination with HSQC, HMBC (see Table 1). Thus, the structure of Compound 1 was identified as 5,7,4' -trihydroxyflavone 7-O-. Beta. -D-glucuronide-6 "-methyl ester, a novel compound not reported in the literature, designated as Dysosma versipellis A (dysosmaflavonoid A).
TABLE 1 Compounds 1 1 H NMR 13 C NMR data (DMSO-d) 6 )
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Compound 2 was a yellow amorphous powder. HR-ESI-MS gives an excimer ion peak M/z: [ M+H ]] + 547.1813 (calculated as 547.1810), molecular formula C was determined 27 H 30 O 12 The unsaturation was 13. Infrared spectra showed the presence of carbonyl groups (1655 cm) -1 ) Hydroxy (3350 cm) -1 ) Benzene ring (1602, 1495 cm) -1 ). The ultraviolet spectrum gives a characteristic maximum absorption of flavonols of 255nm, 336nm. 1 H NMR(500MHz,DMSO-d 6 ) In delta H 6.65 (1 h, d, j=2.1 Hz) and δ H 6.45 (1 h, d, j=2.1 Hz) is meta-coupled, i.eThe characteristic hydrogen signal of the A ring of the flavonol is used for prompting 5, 7-dioxygen substitution. Delta H 6.77 (1 h, d, j=8.0 Hz) and δ H 6.79 (1 h, d, j=8.0 Hz) is ortho-coupled, i.e. characteristic hydrogen signal of flavonol B ring, suggesting 2',3',4' -trisubstituted of B ring. Methoxy signal delta H 3.58 (3H, s). Isopentenyl signal delta H 1.32 (3 h, s), 1.46 (3 h, s), 5.01 (1 h, d, j=5.8 Hz), 3.29 (2 h, d, j=5.8 Hz). Delta 5.02 (1 h, D, j=7.4 Hz) is the terminal proton signal of glucose, the relative configuration is determined to be beta-type according to the coupling constant of the terminal protons, and the absolute configuration of glucose is shown to be D-type by acid hydrolysis and HPLC analysis. 13 C NMR(125MHz，DMSO-d 6 ) Gives 27 carbon signals in total, and shows an isopentenyl signal delta by combining HSQC map data C 25.8,122.8,130.5,17.6,25.5; group of beta-glucosyl carbon signals delta C 99.9, 73.2, 76.5, 69.6, 77.2, 60.7; methoxy delta C 60.0; a flavonol skeleton, wherein delta C 178.4 is the characteristic signal of the carbonyl group at the 4-position of flavonol. In the HMBC pattern, the beta-glucose end group hydrogen proton signal delta H 5.02(1H,d,J＝7.4Hz,δ C 99.9 Delta) and delta C 163.1 The presence of a remote correlation of the carbon signal at (C-7) indicates that the hydroxyl group at position 7 is glycosylated; aromatic methoxy proton signals delta 3.58 (3H, s) and delta C 139.1 The presence of a remote correlation of the carbon signal at (C-3) indicates that the hydroxyl group at position 3 is methylated. Delta from the isopentenyl signal H 3.29 (2 h, d, j=5.8 Hz) and δ C 120.9 The presence of remote correlations of the three carbon signals (C-1 '), 128.0 (C-2'), 143.4 (C-3 '), indicated that the isopentenyl signal was attached to the 2' position of the B-ring of flavonols. According to 1 H-NMR 13 C-NMR, data were specifically assigned in combination with HSQC, HMBC (see Table 2). Thus, the structure of compound 2 was identified as 2' - (3-methyl-2-butenyl) -5,3',4' -trihydroxy-3-methoxyflavone 7-O-beta-D-glucoside, a novel compound not reported in the literature, designated as dysosma versipellis B (dysosmaflavonoid B).
TABLE 2 Compounds 2 1 H NMR 13 C NMR data (DMSO-d) 6 )
Compound 3 was a yellow amorphous powder. HR-ESI-MS gives an excimer ion peak M/z: [ M+H ]] + 547.1804 (calculated as 547.1810), molecular formula C was determined 27 H 30 O 12 The unsaturation was 13. Infrared spectra showed the presence of carbonyl groups (1653 cm) -1 ) Hydroxy (3367 cm) -1 ) Benzene ring (1608, 1500 cm) -1 ). The ultraviolet spectrum gives the characteristic maximum absorption of flavonols at 249nm, 320nm. 1 H NMR(500MHz,DMSO-d 6 ) In delta H 6.75 (1 h, d, j=2.1 Hz) and δ H 6.48 (1 h, d, j=2.1 Hz) is meta-coupled, i.e. the characteristic hydrogen signal of the flavonol a ring, suggesting a 5, 7-dioxygen substitution. Delta H 6.73 (1 h, d, j=8.4 Hz) and δ H 6.74 (1 h, d, j=8.4 Hz) is ortho-coupled, i.e. characteristic hydrogen signal of flavonol B ring, suggesting 2',3',4' -trisubstituted of B ring. Methoxy signal delta H 3.52 (3H, s). Isopentenyl signal delta H 1.33 (3 h, s), 1.49 (3 h, s), 5.04 (1 h, t, j=6.9 Hz), 3.24 (2 h, j=6.9 Hz). Delta 4.76 (1 h, D, j=7.5 Hz) is the terminal proton signal of glucose, the relative configuration is determined to be beta-type according to the coupling constant of the terminal protons, and the absolute configuration of glucose is shown to be D-type by acid hydrolysis and HPLC analysis. 13 C NMR(125MHz，DMSO-d 6 ) Gives 27 carbon signals in total, and shows an isopentenyl signal delta by combining HSQC map data C 25.9, 122.9, 130.0, 17.5, 25.4; group of beta-glucose carbon signals delta C 104.1, 73.6, 75.7, 69.7, 77.6, 60.8; aromatic methoxy delta C 59.5; a flavonol skeleton, wherein delta C 173.3 is the characteristic signal of the carbonyl group at the 4-position of flavonol. In HMBC profile, beta-glucose end group hydrogen proton signals δ4.76 (1 h, d, j=7.5 Hz) and δ C 158.8 The presence of a remote correlation of the carbon signal at (C-5) suggests that the hydroxyl group at position 5 is glycosylated. Methoxy proton signals δ3.52 (3H, s) and δ C 140.6(C-3) The carbon signal at the site is remotely related, indicating that the 3-hydroxy group is methylated. Delta from the isopentenyl signal H 3.24 (2h, d, j=6.9 Hz) and δ C 121.3 The carbon signals at (C-1 '), 127.7 (C-2'), 143.4 (C-3 ') are correlated remotely, indicating that the isopentenyl signal is attached to the 2' position of the flavonol B ring. According to 1 H-NMR 13 C-NMR, data were specifically assigned in combination with HSQC, HMBC (see Table 3). Thus, the structure of compound 3 was identified as 2' - (3-methyl-2-butenyl) -7,3',4' -trihydroxy-3-methoxyflavone 5-O-beta-D-glucoside, a novel compound not reported in the literature, designated as dysosma versipellis C (dysosmaflavonoid C).
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TABLE 3 Compounds 3 1 H NMR 13 C NMR data (DMSO-d) 6 )
Compound 4 was a yellow amorphous powder. HR-ESI-MS gives an excimer ion peak M/z: [ M+H ]] + 547.1814 (calculated as 547.1810), molecular formula C was determined 27 H 30 O 12 The unsaturation was 13. Infrared spectra showed the presence of carbonyl groups (1656 cm) -1 ) Hydroxy (3389 cm) -1 ) Benzene ring (1603, 1487 cm) -1 ). The ultraviolet spectrum gives a characteristic maximum absorption of flavonols of 257nm, 329nm. 1 H NMR(500MHz,DMSO-d 6 ) In delta H 6.18 (1 h, d, j=1.8 Hz) and δ H 6.27 (1 h, d, j=1.8 Hz) is meta-coupling, i.e. a characteristic hydrogen signal of the flavonol a ring, suggesting a 5, 7-dihydroxy substitution on the a ring. Delta H 7.11 (1 h, d, j=8.5 Hz) and δ H 6.88 (1 h, d, j=8.5 Hz) is ortho-coupled, i.e. characteristic hydrogen signal of the flavonol B ring, suggesting 2',3',4' -trisubstituted of the B ring. Methoxy signal delta H 3.59 (3H, s). Isopentenyl signal delta H 1.37(3H,s),1.49(3H,s),5.04(1H,t,J＝6.9Hz),3.29(2H,d,J＝6.9Hz)。 13 C NMR(125MHz，DMSO-d 6 ) Gives 27 carbon signals in total, and shows an isopentenyl carbon signal delta by combining HSQC spectrum data C 25.9, 122.4, 130.6, 17.5, 25.3; group of beta-glucose carbon signals delta C 102.2, 73.3, 75.6, 69.9, 77.3, 60.8; aromatic methoxy delta C 60.0; a flavonol skeleton, wherein delta C 177.9 is a characteristic signal of the carbonyl group at the 4-position of flavonol. δ4.76 (1 h, d, j=7.4 Hz) is the terminal proton signal of the sugar, and the glycosidic bond is determined to be β -configuration according to the coupling constant of its terminal proton. In HMBC profile, methoxy proton signal delta H 3.59 (3H, s) and delta C 138.8 The presence of a remote correlation of the carbon signal at (C-3) indicates that the hydroxyl group at position 3 is methylated. Beta-glucose end group hydrogen proton signals δ4.76 (1 h, d, j=7.4 Hz) and δ C 146.7 The presence of a remote correlation of the carbon signal at (C-4 ') suggests that the hydroxyl group at the 4' position is glycosylated. Delta from the isopentenyl signal H 3.29 (2h, d, j=6.9 Hz) and δ C 124.3 The carbon signals at (C-1 '), 127.6 (C-2'), 144.6 (C-3 ') are correlated remotely, indicating that the isopentenyl signal is attached to the 2' position of the B-ring of flavonols. According to 1 H-NMR 13 C-NMR, data were specifically assigned in combination with HSQC, HMBC (see Table 4). Thus, the structure of compound 4 was identified as 2' - (3-methyl-2-butenyl) -5,7,3' -trihydroxy-3-methoxyflavone 4' -O-beta-D-glucoside, a novel compound not reported in the literature, designated as dysosma versipellis D (dysosmaflavonoid D).
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TABLE 4 Compounds 4 1 H NMR 13 C NMR data (DMSO-d) 6 )
Compound 5 was a yellow amorphous powder. HR-ESI-MS gives an excimer ion peak M/z: [ M+H ]] + 499.1234 (calculated as 499.1234), molecular formula C was determined 25 H 22 O 11 The degree of unsaturation was 15. Infrared spectra showed the presence of carbonyl groups (1652 cm) -1 ) Hydroxyl (3368 cm) -1 ) Benzene ring (1602, 1516 cm) -1 ). The ultraviolet spectrum gives a characteristic maximum absorption of flavonols of 251nm, 320nm. 1 H NMR(500MHz,DMSO-d 6 ) In delta H 12.54 (1H, s) is a characteristic signal of the phenolic hydroxyl group at the 5-position of flavonol. The aromatic zone gives characteristic hydrogen signals of benzene rings of two groups of ABX coupling systems, delta respectively H 7.73(1H,s)、6.90(1H,d,J＝8.4Hz)、7.58(1H,d,J＝8.4Hz)；δ H 7.03 (1 h, s), 6.61 (1 h, d, j=8.1 Hz), 6.68 (1 h, d, j=8.1 Hz), one of which is a characteristic hydrogen signal of the B-ring of the flavonol mother nucleus. Delta H 6.22 (1H, s) is an aromatic hydrogen proton signal and is a characteristic hydrogen signal of the 6-position of a flavonol A ring. Delta H 4.88 (1 h, d, j=5.5 Hz), 4.48 (1 h, q, j=5.5 Hz), 3.24 (2 h, m) is 2, 3-dihydroxypropyl; methoxy signal delta H 3.66(3H,s)。 13 C NMR(125MHz，DMSO-d 6 ) And DEPT pattern gives 27 carbon signals, an aromatic methoxy delta C 55.4,1 benzene rings, 12, 3-dihydroxypropyl delta C 42.6, 73.3, 64.1, a flavonol backbone carbon signal, wherein δ C 176.1 is a characteristic signal of the carbonyl group at the 4-position of flavonol. In HMBC profile, δ H 4.88 (1 h, d, j=5.5 Hz) is remotely correlated with ABX coupled benzene ring carbon signals 132.6 (C-1 "), 112.6 (C-2"), 120.4 (C-6 "), suggesting the presence of 1 '" - (3 ", 4" -dihydroxyphenyl) -2 ' ", 3 '" -hydroxy-propyl. From delta H 4.88 (1 h, d, j=5.5 Hz) flavonol parent nucleus δ C 162.5 (C-7), 106.2 (C-8), 154.1 (C-9) and the 8-position of the flavonol A ring are remotely related to 1 ' - (3 ', 4' -dihydroxyphenyl) -2 ', 3' -hydroxy-propyl groups are directly linked. Aromatic methoxy proton signal delta H 3.66 (3H, s) and delta C 147.2 The carbon signal at (C-4 ') is remotely correlated, indicating that the methoxy group is attached at the C-4' position. According to 1 H-NMR 13 C-NMR, data were specifically assigned in combination with HSQC, HMBC (see Table 5). The absolute configuration of the C-1 '"and C-2'" positions was determined by ECD spectral data analysis, and the ECD spectral data tested and calculated ECD spectral numbers of the isomer (1 '"S, 2'" R) -5The absolute configuration of the C-1 '", C-2'" positions of compound 5 was determined to be 1 '"S, 2'" R, respectively, according to substantial agreement. Thus, the structure of Compound 5 was identified as (1 ' "S, 2 '" R) -8- [1 ' "- (3", 4 "-dihydroxy-phenyl) -2 '", 3' "-dihydroxy-propyl]3,5,7,3'-tetrahydroxy-4' -methoxyflavone is a novel compound which is not reported in the literature and is named as dysosma versipellis E (dysosmaflavonoid E).
TABLE 5 Compounds 5 1 H NMR 13 C NMR data (DMSO-d) 6 )
Compound 6 was a yellow amorphous powder. HR-ESI-MS gives an excimer ion peak M/z: [ M+H ]] + 453.0809 (calculated as 453.0822), molecular formula C was determined 23 H 16 O 10 The unsaturation was 16. Infrared spectra showed the presence of carbonyl groups (1652 cm) -1 ) Hydroxy (3402 cm) -1 ) Benzene ring (1608, 1513 cm) -1 ). The ultraviolet spectrum gives a characteristic maximum absorption of flavonols of 264nm, 370nm. 1 H NMR(500MHz,DMSO-d 6 ) In delta H 12.49 (1H, s) is a characteristic signal of the phenolic hydroxyl group at the 5-position of flavonol. The aromatic region gives a 1, 4-disubstituted benzene ring hydrogen signal, delta H 7.63 (2h, d, j=8.0 Hz), 6.72 (2h, d, j=8.0 Hz). 1,2,3, 4-tetrasubstituted benzene ring hydrogen signal delta H 7.38 (1 h, d, j=8.5 Hz), 6.90 (1 h, d, j=8.5 Hz). Penta-substituted benzene ring hydrogen signal delta H 6.27 (1H, d, s). Methoxy hydrogen signal delta H 3.43(3H,s)。 13 C NMR(125MHz，DMSO-d 6 ) The spectrum gives 23 carbon signals, namely methoxy delta C 51.2, an ester carbonyl carbon Signal delta C 166.6,1 benzene rings, a flavonol backbone carbon signal, wherein delta C 176.2 is a characteristic signal of the carbonyl group at the 4-position of flavonol. By comparison with the literature, flavonol knotsThe building block was identified as kaempferol. From methoxy hydrogen signal and ester carbonyl carbon signal delta C 166.6, determining the presence of 2, 3-dihydroxy-6-methoxycarbonyl-phenyl. The 2, 3-dihydroxy-6-methoxycarbonyl-phenyl group was suggested to be attached to C-8 of kaempferol by a change in the chemical shift values from δc165.5 (C-7), 94.5 (C-8), 158.2 (C-9) to δc 161.2 (C-7), 102.9 (C-8), 153.2 (C-9) in compound 6. According to 1 H-NMR 13 C-NMR, data were specifically assigned in combination with HSQC, HMBC (see Table 6). Thus, the structure of Compound 6 was identified as 8- (2 ', 3' -dihydroxy-6 '-methoxycarbonyl-phenyl) -3,5,7,4' -tetrahydroflavone, a novel compound not reported in the literature, designated Dysosma versipellis F (dysosmaflavonoid F).
TABLE 6 Compounds 6 1 H NMR 13 C NMR data (DMSO-d) 6 )
2. Antioxidant Activity
DPPH solution (100 mu M final concentration) and the sample solution to be tested (6.25,12.5,25,50,100 mu M final concentration) with different concentrations were mixed well, and the mixture was left to stand in the dark at room temperature for 1 hour, and the absorbance was measured at 515 nm. IC for DPPH free radical scavenging ability 50 The value represents. The results are shown in Table 7.
TABLE 7 free radical scavenging ability of Compounds 1-6 against DPPH
The experiments show that the novel flavonol compounds prepared by the invention, including dysosma versipellis A (dysosmaflavonoid A), dysosma versipellis B (dysosmaflavonoid B), dysosma versipellis C (dysosmaflavonoid C), dysosma versipellis E (dysosmaflavonoid E) and dysosma versipellis F (dysosmaflavonoid F), have the capability of scavenging DPPH free radicals, and can be used as an antioxidant for food or beverage.
Claims (6)
1. The flavonol compounds dysosma versipellis A-C, E-F are dysosma versipellis A, dysosma versipellis B, dysosma versipellis C, dysosma versipellis E and dysosma versipellis F extracted from dysosma versipellis, and the molecular structural formulas are respectively:
2. The method for preparing the flavonol compound dysosma versipellis A-C, E-F as claimed in claim 1, which is characterized by comprising the following steps: 20-40kg of dysosma versipellis medicinal material is taken as a raw material, 3-5 times of ethanol with the weight volume and the volume concentration of 95% is added each time, the ethanol is heated and refluxed for extraction for 3 times, the weight volume is calculated by kg of solid, the liquid is calculated by L, the extraction temperature is 92-95 ℃, the extraction time is 1-1.5 hours each time, and the ethanol with the volume concentration of 95% is recovered under reduced pressure to obtain an extract of 95% ethanol; adding 3-5 times of ethanol with weight volume and volume concentration of 50% into the residue, reflux extracting for 1 time at 92-95deg.C for 1-1.5 hr, and recovering 50% ethanol under reduced pressure to obtain extract of 50% ethanol; mixing 95% ethanol extract and 50% ethanol extract, adding anhydrous ethanol 2 times the weight of the extract for dissolution, adding diatomite 2 times the weight of the extract for adsorption, recovering solvent, sequentially adding dichloromethane, ethyl acetate and methanol 3 times the weight of the extract for elution, and recovering solvent to obtain dichloromethane elution part, ethyl acetate elution part and methanol elution part respectively; separating the methanol elution part by silica gel column chromatography, sequentially performing gradient elution by using a dichloromethane-methanol mixed solvent system with volume ratios of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:30, 100:50 and 0:100, wherein each gradient uses 30L-60L eluent with the flow rate of 50-70mL/min, each 5L-10L is a fraction, and collecting 54 fractions, and each fraction passes through a silica gel thin layer Chromatographic detection analysis using GF 254 The thin layer plate is prepared by respectively taking petroleum ether-acetone with the volume ratio of 4:5 and methylene dichloride-methanol with the volume ratio of 5:2 as developing agents, taking sulfuric acid-ethanol solution with the volume ratio of 10:90 as a developing agent, heating for 3-5min at the temperature of 105 ℃, and respectively combining 1-12 parts, 13-24 parts, 25-30 parts, 31-36 parts, 37-38 parts, 39-42 parts, 43-44 parts, 45-48 parts and 49-54 parts according to the detection result of thin layer chromatography to obtain a component Fr.M1, a component Fr.M2, a component Fr.M3, a component Fr.M4, a component Fr.M5, a component Fr.M6, a component Fr.M7, a component Fr.M8 and a component Fr.M9;
separating the component Fr.M4 by sephadex LH-20 gel column chromatography, eluting with 5.0L-10L of methanol at a flow rate of 5-10mL/min, collecting 50 fractions each 100-200 mL as one fraction, and respectively combining 1-25 fractions, 26-44 fractions and 45-50 fractions by silica gel thin layer chromatography detection analysis to obtain a subfraction Fr.M4-1, a subfraction Fr.M4-2 and a subfraction Fr.M4-3; the subfraction Fr.M4-1 was separated by ODS column chromatography with MeOH-H in a volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, wherein each gradient is 2.5L-5L, each gradient is a subcomponent, and subcomponent Fr.M4-1-1, subcomponent Fr.M4-1-2, subcomponent Fr.M4-1-3, subcomponent Fr.M4-1-4, subcomponent Fr.M4-1-5 and subcomponent Fr.M4-1-6 are obtained; separating the subfractions Fr.M4-1-2 by sephadex LH-20 gel column chromatography, eluting with 500-1000mL of methanol, taking each 5-10mL as a fraction, collecting 100 fractions, respectively combining the fractions 1-17, 18-41, 42-59, 60-77, 78-92 and 93-100 by silica gel thin layer chromatography detection analysis, and collecting the fractions to obtain subfractions Fr.M4-1-2-1, subfractions Fr.M4-1-2-2, subfractions Fr.M4-1-2-3, subfractions Fr.M4-1-2-4, subfractions Fr.M4-1-2-5 and subfractions Fr.M4-1-2-6; purifying the subfraction M4-1-2-3 by preparative high performance liquid chromatography, eluting with mixed methanol-water solvent with volume ratio of 52:48, collecting chromatographic peaks with retention time of 7.9min, 8.3min, 10.8min, 12.8min, 16.4min, and 25.1min respectively, to obtain subfraction Fr.M4-1-2-3-1, subfraction Fr.M4-1-2-3-2, subfraction Fr.M4-1-2-3-3, subfraction Fr.M4-1-2-3-4, subfraction Fr.M4-1-2-3 -5, subfraction fr.m4-1-2-3-6; purifying the subfraction Fr.M4-1-2-3-3 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peaks with retention time of 17.5min respectively, wherein the chromatographic column is YMC-Pack ODS-A, and the flow rate is 3 m/min, to obtain dysosmse:Sup>A versipellis E;
purifying the subfraction Fr.M4-1-2-3-4 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peak with flow rate of 3 m/min and retention time of 30.7min to obtain dysosmse:Sup>A versipellis ketone F;
separating the component Fr.M5 by sephadex LH-20 column chromatography, eluting with 2.5L-5L methanol at a flow rate of 5-10mL/min, collecting 25 fractions each 100mL-200mL as one fraction, and respectively combining 1-3, 4-5, 6-7, 8-10, 11-13, 14-17, 19-21 and 22-25 by silica gel thin layer chromatography detection analysis to obtain a subfraction Fr.M5-1, a subfraction Fr.M5-2, a subfraction Fr.M5-3, a subfraction Fr.M5-4, a subfraction Fr.M5-5, a subfraction Fr.M5-6, a subfraction Fr.M5-7 and a subfraction Fr.M5-8; the subfractions Fr.M5-1 to Fr.M5-5 are combined, separated by MCI column chromatography and separated by MeOH-H with volume ratio of 0:100, 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Carrying out gradient elution on an O mixed solvent system, wherein each gradient elution solvent is 1.0L-2.0L, the flow rate is 2.5-5.0mL/min, each 250mL-500mL is a fraction, 28 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-5 fractions, 6-15 fractions, 16-23 fractions and 17-28 fractions are respectively combined to obtain a subfraction Fr.M5-1, a subfraction Fr.M5-1-2, a subfraction Fr.M5-1-3 and a subfraction Fr.M5-1-4; the subfractions Fr.M5-1-3 were separated by column chromatography on silica gel with CHCl in a volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 7:1, 3:1 3 Gradient elution of MeOH mixed solvent systems, wherein each gradient elution solvent is 50-100 mL, the flow rate is 2.0-5.0mL/min, each 5-10 mL is one fraction, 80 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-10, 11-20, 21-30, 31-40, 41-46, 47-51, 52-60, 61-70 and 71-80 of the fractions are respectively combined to obtain a subfraction Fr.M5-1-3-1, a subfraction Fr.M5-1-3-2 and a subfraction Fr.M5-1-3-2M5-1-3-3, subfraction Fr.M5-1-3-4, subfraction Fr.M5-1-3-5, subfraction Fr.M5-1-3-6, subfraction Fr.M5-1-3-7, subfraction Fr.M5-1-3-8, subfraction Fr.M5-1-3-9; purifying the subfraction Fr.M5-1-3-5 by preparative high performance liquid chromatography, eluting with methanol-water mixed solvent with volume ratio of 55:45, collecting chromatographic peak with retention time of 34.9min at flow rate of 3 m/min with YMC-Pack ODS-A column to obtain dysosmse:Sup>A versipellis ketone A;
Separating the component Fr.M6 by sephadex LH-20 column chromatography, eluting with 15L-30L methanol at a flow rate of 5-10mL/min, collecting 30 fractions each 0.5L-1L as one fraction, and respectively combining fractions 1-18 and 19-30 by silica gel thin layer chromatography detection analysis to obtain subfractions Fr.M6-1 and Fr.M6-2; the subfraction Fr.M6-1 is separated by ODS column chromatography with MeOH-H in volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out on the O mixed solvent system, wherein each gradient elution solvent is 2.5L-5L, the flow rate is 3.0-6.0mL/min, each 0.5L-1L is one fraction, 30 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-5 fractions, 6-10 fractions, 11-20 fractions and 21-30 fractions are respectively combined to obtain a subfraction Fr.M6-1-1, a subfraction Fr.M6-1-2, a subfraction Fr.M6-1-3 and a subfraction M6-1-4; separating the subfractions Fr.M6-1-3 by silica gel column chromatography, and performing gradient elution by using a methylene dichloride-methanol mixed solvent system with the volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10 and 100:30, wherein each gradient elution solvent is 125L-250L, the flow rate is 1.0-2.0mL/min, and each elution gradient is one flow part, so as to obtain subfractions Fr.M6-1-3-1, subfractions Fr.M6-1-3-2, subfractions Fr.M6-1-3-3, subfractions Fr.M6-1-3-4, subfractions Fr.M6-1-3-5, subfractions Fr.M6-1-3-6 and subfractions Fr.M6-1-3-7; separating the subfractions Fr.M6-1-3-4 by sephadex LH-20 gel column chromatography, eluting with 125mL-250mL of methanol at a flow rate of 1-2mL/min, collecting 25 fractions each of which is 5mL-10mL, and respectively combining the fractions 1-7, 8-16 and 17-25 by silica gel thin layer chromatography detection analysis to obtain subfractions Fr.M6-1-3-4-1, subfractions Fr.M6-1-3-4-2 and subfractions Fr.M6-1-3-4-3; purifying the subfraction Fr.M6-1-3-4-2 by preparative high performance liquid chromatography, eluting with 50:50 methanol-water mixed solvent, and collecting the eluate with se:Sup>A flow rate of 3, wherein the chromatographic column is YMC-Pack ODS-A Collecting chromatographic peaks with retention time of 33.1min, 41.5min and 45.0min respectively at mL/min to obtain subfraction Fr.M6-1-3-4-2-1, subfraction Fr.M6-1-3-4-2-2 and subfraction Fr.M6-1-3-4-2-3; purifying the subfraction Fr.M6-1-3-4-2-1 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 35:65, collecting chromatographic peak with flow rate of 3 m/min and retention time of 11.9min to obtain compound dysosmse:Sup>A versipellis C;
purifying the subfraction Fr.M6-1-3-4-2-3 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 30:70, collecting chromatographic peak with flow rate of 3 m/min and retention time of 28.5min, and collecting the compound dysosmse:Sup>A versipellis ketone B.
3. The method for preparing the flavonol compound dysosma versipellis ketone A-C, E-F according to claim 2, wherein 40kg of dysosma versipellis medicinal material is taken as a raw material, 3 times of ethanol with the weight, volume and volume concentration of 95% is added into the raw material each time, the extraction temperature is 95 ℃ and the extraction time is 1 hour, and 95% ethanol is recovered under reduced pressure to obtain an extract of 95% ethanol; adding 3 times of ethanol with weight volume and volume concentration of 50% into the residue, reflux extracting for 1 time at 95deg.C for 1 hr, and recovering 50% ethanol under reduced pressure to obtain extract of 50% ethanol; mixing 95% ethanol extract and 50% ethanol extract, adding 10.8L of absolute ethanol for dissolution, adding 10.8kg of diatomite for adsorption, recovering solvent, sequentially adding 16.2L of dichloromethane, ethyl acetate and methanol for eluting, and recovering solvent to obtain dichloromethane eluting part, ethyl acetate eluting part and methanol eluting part respectively; separating the methanol elution part by silica gel column chromatography, sequentially performing gradient elution by using a dichloromethane-methanol mixed solvent system with volume ratios of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:30, 100:50 and 0:100, wherein each gradient uses 60L eluent with the flow rate of 70mL/min and each 10L is one fraction, collecting 54 fractions, detecting and analyzing each fraction by silica gel thin layer chromatography, and using GF 254 The thin layer plate is prepared from petroleum ether-acetone in a volume ratio of 4:5 and two of petroleum ether-acetone in a volume ratio of 5:2 respectivelyMethyl chloride-methanol is used as developing agent, sulfuric acid-ethanol solution with the volume ratio of 10:90 is used as color developing agent, heating is carried out for 4min at 105 ℃, and according to the detection result of thin layer chromatography, 1-12 parts of flow, 13-24 parts of flow, 25-30 parts of flow, 31-36 parts of flow, 37-38 parts of flow, 39-42 parts of flow, 43-44 parts of flow, 45-48 parts of flow and 49-54 parts of flow are respectively combined to obtain a component Fr.M1, a component Fr.M2, a component Fr.M3, a component Fr.M4, a component Fr.M5, a component Fr.M6, a component Fr.M7, a component Fr.M8 and a component Fr.M9;
separating the component Fr.M4 by sephadex LH-20 gel column chromatography, eluting with 10L of methanol at a flow rate of 10mL/min, collecting 50 fractions each 200mL as one fraction, and respectively combining fractions 1-25, 26-44 and 45-50 by silica gel thin layer chromatography detection analysis to obtain a subfraction Fr.M4-1, a subfraction Fr.M4-2 and a subfraction Fr.M4-3; the subfraction Fr.M4-1 was separated by ODS column chromatography with MeOH-H in a volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, wherein each gradient is 5L, and each gradient is a subcomponent, so that subcomponent Fr.M4-1-1, subcomponent Fr.M4-1-2, subcomponent Fr.M4-1-3, subcomponent Fr.M4-1-4, subcomponent Fr.M4-1-5 and subcomponent Fr.M4-1-6 are obtained; separating the subfractions Fr.M4-1-2 by sephadex LH-20 gel column chromatography, eluting with 1000mL of methanol, taking the flow rate as 5mL/min, taking 10mL as one fraction, collecting 100 fractions, respectively combining 1-17, 18-41, 42-59, 60-77, 78-92 and 93-100 of the fractions by silica gel thin layer chromatography detection analysis, and collecting the subfractions Fr.M4-1-2-1, fr.M4-1-2, fr.M4-2-3, fr.M4-1-2-4, fr.M4-1-2-5 and Fr.M4-1-2-6; purifying the subfraction M4-1-2-3 by preparative high performance liquid chromatography, eluting with se:Sup>A mixed solvent of methanol and water with se:Sup>A volume ratio of 52:48, collecting chromatographic peaks with retention time of 7.9min, 8.3min, 10.8min, 12.8min, 16.4min and 25.1min respectively for the chromatographic column YMC-Pack ODS-A, the flow rate of 3 m/min, and obtaining subfraction Fr.M4-1-2-3-1, subfraction Fr.M4-1-2-3-2, subfraction Fr.M4-1-2-3-3, subfraction Fr.M4-1-2-3-4, subfraction Fr.M4-1-2-3-5 and subfraction Fr.M4-1-2-3-6; the subfraction Fr.M4-1-2-3-3 is purified by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with the volume ratio of 27:73, The chromatographic column is YMC-Pack ODS-A, the flow rate is 3 m/min, and chromatographic peaks with retention time of 17.5min are respectively collected to obtain dysosmse:Sup>A versipellis E;
purifying the subfraction Fr.M4-1-2-3-4 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peak with flow rate of 3 m/min and retention time of 30.7min to obtain dysosmse:Sup>A versipellis ketone F;
separating the component Fr.M5 by sephadex LH-20 column chromatography, eluting with 5L of methanol, wherein the flow rate is 10mL/min, taking 200mL as one fraction, collecting 25 fractions, respectively combining 1-3 fractions, 4-5 fractions, 6-7 fractions, 8-10 fractions, 11-13 fractions, 14-17 fractions, 19-21 fractions and 22-25 fractions by silica gel thin layer chromatography detection analysis to obtain a subfraction Fr.M5-1, a subfraction Fr.M5-2, a subfraction Fr.M5-3, a subfraction Fr.M5-4, a subfraction Fr.M5-5, a subfraction Fr.M5-6, a subfraction Fr.M5-7 and a subfraction Fr.M5-8; the subfractions Fr.M5-1 to Fr.M5-5 are combined, separated by MCI column chromatography and separated by MeOH-H with volume ratio of 0:100, 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Carrying out gradient elution on an O mixed solvent system, wherein each gradient elution solvent is 2.0L, the flow rate is 5.0mL/min, each 500mL is a fraction, 28 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-5, 6-15, 16-23 and 17-28 of the fractions are respectively combined to obtain a subfraction Fr.M5-1, a subfraction Fr.M5-1-2, a subfraction Fr.M5-1-3 and a subfraction Fr.M5-1-4; the subfractions Fr.M5-1-3 were separated by column chromatography on silica gel with CHCl in a volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 7:1, 3:1 3 Gradient elution is carried out by a MeOH mixed solvent system, wherein each gradient elution solvent is 100mL, the flow rate is 5.0mL/min, each 10mL is one fraction, 80 fractions are collected, each fraction is subjected to silica gel thin layer chromatography detection analysis, and 1-10 fractions, 11-20 fractions, 21-30 fractions, 31-40 fractions, 41-46 fractions, 47-51 fractions, 52-60 fractions, 61-70 fractions and 71-80 fractions are respectively combined to obtain subfractions Fr.M5-1-3-1, subfractions Fr.M5-1-3-2, subfractions Fr.M5-1-3-3, subfractions Fr.M5-1-4, subfractions Fr.M5-1-3-5, subfractions Fr.M5-1-3-6, subfractions Fr.M5-1-3-8, subfractions Fr.M5-1-3-9; subfraction Fr.M5-1-3-5 is prepared with high efficiencyPurifying by liquid chromatography, eluting with methanol-water mixed solvent with volume ratio of 55:45, collecting chromatographic peak with retention time of 34.9min at flow rate of 3 m/min with YMC-Pack ODS-A to obtain dysosmse:Sup>A versipellis ketone A;
separating the component Fr.M6 by sephadex LH-20 column chromatography, eluting with 30L of methanol at a flow rate of 10mL/min, collecting 30 fractions each 1L as one fraction, and respectively combining fractions 1-18 and 19-30 by silica gel thin layer chromatography detection analysis to obtain sub-components Fr.M6-1 and Fr.M6-2; the subfraction Fr.M6-1 is separated by ODS column chromatography with MeOH-H in volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, wherein each gradient elution solvent is 5L, the flow rate is 6.0mL/min, each 1L is one fraction, 30 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-5 fractions, 6-10 fractions, 11-20 fractions and 21-30 fractions are respectively combined to obtain a subfraction Fr.M6-1-1, a subfraction Fr.M6-1-2, a subfraction Fr.M6-1-3 and a subfraction M6-1-4; separating the subfractions Fr.M6-1-3 by silica gel column chromatography, and performing gradient elution by using a methylene dichloride-methanol mixed solvent system with the volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10 and 100:30, wherein each gradient elution solvent is 250L, the flow rate is 2.0mL/min, and each elution gradient is a flow part, so as to obtain subfractions Fr.M6-1, subfractions Fr.M6-1-3-2, subfractions Fr.M6-1-3-3, subfractions Fr.M6-1-3-4, subfractions Fr.M6-1-3-5, subfractions Fr.M6-1-3-6 and subfractions Fr.M6-1-3-7; separating the subfractions Fr.M6-1-3-4 by sephadex LH-20 gel column chromatography, eluting with 250mL of methanol, wherein the flow rate is 2mL/min, taking 10mL as one fraction, collecting 25 fractions, respectively combining the fractions 1-7, 8-16 and 17-25 by silica gel thin layer chromatography detection analysis to obtain subfractions Fr.M6-1-3-4-1, subfractions Fr.M6-1-3-4-2 and subfractions Fr.M6-1-3-4-3; purifying the subfraction Fr.M6-1-3-4-2 by preparative high performance liquid chromatography, eluting with methanol-water mixed solvent with volume ratio of 50:50, collecting chromatographic peaks with retention time of 33.1min, 41.5min and 45.0min respectively for the chromatographic column YMC-Pack ODS-A and flow rate of 3 m/min, and obtaining subfraction Fr.M6-1-3-4-2-1, subfraction Fr.M6-1-3-4-2-2 and subfraction Fr.M6-1-3-4-2-3; the subfraction Fr.M6-1-3-4-2-1 is purified by preparative high performance liquid chromatography to obtain the body Eluting with acetonitrile-water mixed solvent with volume ratio of 35:65, collecting chromatographic peak with retention time of 11.9min at flow rate of 3 m/min with YMC-Pack ODS-A chromatography column to obtain compound dysosmse:Sup>A versipellis ketone C;
purifying the subfraction Fr.M6-1-3-4-2-3 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 30:70, collecting chromatographic peak with flow rate of 3 m/min and retention time of 28.5min, and collecting the compound dysosmse:Sup>A versipellis ketone B.
4. The method for preparing the flavonol compound dysosma versipellis ketone A-C, E-F according to claim 2, wherein 20kg of dysosma versipellis medicinal material is taken as a raw material, ethanol with the weight, volume and volume concentration of 95% is added into the raw material for each time, the heating reflux extraction is carried out for 3 times, the extraction temperature is 92 ℃, the extraction time is 1.5 hours each time, and 95% ethanol is recovered under reduced pressure to obtain an extract of 95% ethanol; adding ethanol with weight volume of 5 times and volume concentration of 50% into the residue, reflux extracting for 1 time at 92 deg.C for 1.5 hr, and recovering 50% ethanol under reduced pressure to obtain extract 50% ethanol extract; mixing 95% ethanol extract and 50% ethanol extract, adding 5.4L absolute ethanol for dissolving, adding 5.4kg diatomite for adsorption, recovering solvent, sequentially adding 8.1L dichloromethane, ethyl acetate and methanol for eluting, and recovering solvent to obtain dichloromethane eluting part, ethyl acetate eluting part and methanol eluting part respectively; separating the methanol elution part by silica gel column chromatography, sequentially performing gradient elution by using a dichloromethane-methanol mixed solvent system with volume ratios of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:30, 100:50 and 0:100, wherein each gradient uses 30L of eluent, the flow rate is 50mL/min, each 5L is one fraction, 54 fractions are collected, each fraction is detected and analyzed by silica gel thin layer chromatography, and GF is used for detecting and analyzing the components by using GF 254 The thin layer plate takes petroleum ether-acetone with the volume ratio of 4:5 and dichloromethane-methanol with the volume ratio of 5:2 as developing agents, takes sulfuric acid-ethanol solution with the volume ratio of 10:90 as a color developing agent, is heated for 5min at the temperature of 105 ℃, and respectively combines the components of 1-12, 13-24, 25-30, 31-36, 37-38, 39-42,43-44 parts, 45-48 parts and 49-54 parts to obtain a component Fr.M1, a component Fr.M2, a component Fr.M3, a component Fr.M4, a component Fr.M5, a component Fr.M6, a component Fr.M7, a component Fr.M8 and a component Fr.M9;
separating the component Fr.M4 by sephadex LH-20 gel column chromatography, eluting with 5.0L of methanol at a flow rate of 5mL/min, collecting 50 fractions per 100mL, and respectively combining fractions 1-25, 26-44 and 45-50 by silica gel thin layer chromatography detection analysis to obtain a subfraction Fr.M4-1, subfraction Fr.M4-2 and subfraction Fr.M4-3; the subfraction Fr.M4-1 was separated by ODS column chromatography with MeOH-H in a volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, wherein each gradient is 2.5L, each gradient is a subcomponent, and subcomponent Fr.M4-1-1, subcomponent Fr.M4-1-2, subcomponent Fr.M4-1-3, subcomponent Fr.M4-1-4, subcomponent Fr.M4-1-5 and subcomponent Fr.M4-1-6 are obtained; separating the subfractions Fr.M4-1-2 by sephadex LH-20 gel column chromatography, eluting with 500mL of methanol, taking each 5mL as a fraction, collecting 100 fractions, respectively combining 1-17, 18-41, 42-59, 60-77, 78-92 and 93-100 by silica gel thin layer chromatography detection analysis, and collecting the subfractions Fr.M4-1-2-1, fr.M4-1-2-2, fr.M4-1-2-3, fr.M4-1-2-4, fr.M4-1-2-5 and Fr.M4-1-2-6; purifying the subfraction M4-1-2-3 by preparative high performance liquid chromatography, eluting with se:Sup>A mixed solvent of methanol and water with se:Sup>A volume ratio of 52:48, collecting chromatographic peaks with retention time of 7.9min, 8.3min, 10.8min, 12.8min, 16.4min and 25.1min respectively for the chromatographic column YMC-Pack ODS-A, the flow rate of 3 m/min, and obtaining subfraction Fr.M4-1-2-3-1, subfraction Fr.M4-1-2-3-2, subfraction Fr.M4-1-2-3-3, subfraction Fr.M4-1-2-3-4, subfraction Fr.M4-1-2-3-5 and subfraction Fr.M4-1-2-3-6; purifying the subfraction Fr.M4-1-2-3-3 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peaks with retention time of 17.5min respectively, wherein the chromatographic column is YMC-Pack ODS-A, and the flow rate is 3 m/min, to obtain dysosmse:Sup>A versipellis E;
Purifying the subfraction Fr.M4-1-2-3-4 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peak with flow rate of 3 m/min and retention time of 30.7min to obtain dysosmse:Sup>A versipellis ketone F;
separating the component Fr.M5 by sephadex LH-20 column chromatography, eluting with 2.5L of methanol, wherein the flow rate is 5mL/min, taking each 100mL as one fraction, collecting 25 fractions, respectively combining 1-3 fractions, 4-5 fractions, 6-7 fractions, 8-10 fractions, 11-13 fractions, 14-17 fractions, 19-21 fractions and 22-25 fractions by silica gel thin layer chromatography detection analysis to obtain a subfraction Fr.M5-1, a subfraction Fr.M5-2, a subfraction Fr.M5-3, a subfraction Fr.M5-4, a subfraction Fr.M5-5, a subfraction Fr.M5-6, a subfraction Fr.M5-7 and a subfraction Fr.M5-8; the subfractions Fr.M5-1 to Fr.M5-5 are combined, separated by MCI column chromatography and separated by MeOH-H with volume ratio of 0:100, 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Carrying out gradient elution on an O mixed solvent system, wherein each gradient elution solvent is 1.0L, the flow rate is 2.5mL/min, each 250mL is one fraction, 28 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-5, 6-15, 16-23 and 17-28 of the fractions are respectively combined to obtain a subfraction Fr.M5-1, a subfraction Fr.M5-1-2, a subfraction Fr.M5-1-3 and a subfraction Fr.M5-1-4; the subfractions Fr.M5-1-3 were separated by column chromatography on silica gel with CHCl in a volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 7:1, 3:1 3 Gradient elution is carried out by a MeOH mixed solvent system, each gradient elution solvent is 50mL, the flow rate is 2.0mL/min, each 5mL is one fraction, 80 fractions are collected, each fraction is subjected to silica gel thin layer chromatography detection analysis, and 1-10, 11-20, 21-30, 31-40, 41-46, 47-51, 52-60, 61-70 and 71-80 of the fractions are respectively combined to obtain subfractions Fr.M5-1-3-1, subfractions Fr.M5-1-3-2, subfractions Fr.M5-1-3-3, subfractions Fr.M5-1-3-4, subfractions Fr.M5-1-3-5, subfractions Fr.M5-1-3-6, subfractions Fr.M5-1-3-7, subfractions Fr.M5-1-3-8 and subfractions Fr.M5-1-3-9; purifying the subfraction Fr.M5-1-3-5 by preparative high performance liquid chromatography, eluting with methanol-water mixed solvent with volume ratio of 55:45, collecting chromatographic peak with retention time of 34.9min at flow rate of 3 m/min with YMC-Pack ODS-A column to obtain dysosmse:Sup>A versipellis ketone A;
component Fr.M6 warpsephadex LH-20 column chromatography, eluting with 15L methanol at a flow rate of 5mL/min, collecting 30 fractions every 0.5L, and subjecting each fraction to silica gel thin layer chromatography detection analysis to obtain subfractions Fr.M6-1 and Fr.M6-2 by respectively combining fractions 1-18 and 19-30; the subfraction Fr.M6-1 is separated by ODS column chromatography with MeOH-H in volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, each gradient elution solvent is 2.5L, the flow rate is 3.0mL/min, each 0.5L is one fraction, 30 fractions are collected, each fraction is detected and analyzed by silica gel thin layer chromatography, and 1-5 fractions, 6-10 fractions, 11-20 fractions and 21-30 fractions are combined to obtain a subfraction Fr.M6-1-1, a subfraction Fr.M6-1-2, a subfraction Fr.M6-1-3 and a subfraction M6-1-4; separating the subfractions Fr.M6-1-3 by silica gel column chromatography, and performing gradient elution by using a methylene dichloride-methanol mixed solvent system with the volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10 and 100:30, wherein each gradient elution solvent is 125L, the flow rate is 1.0mL/min, and each elution gradient is a flow part, so as to obtain subfractions Fr.M6-1, subfractions Fr.M6-1-3-2, subfractions Fr.M6-1-3-3, subfractions Fr.M6-1-3-4, subfractions Fr.M6-1-3-5, subfractions Fr.M6-1-3-6 and subfractions Fr.M6-1-3-7; separating the subfractions Fr.M6-1-3-4 by sephadex LH-20 gel column chromatography, eluting with 125mL of methanol at a flow rate of 1mL/min, collecting 25 fractions each of which is one fraction per 5mL, and respectively combining the fractions 1-7, 8-16 and 17-25 by silica gel thin layer chromatography detection analysis to obtain subfractions Fr.M6-1-3-4-1, subfractions Fr.M6-1-3-4-2 and subfractions Fr.M6-1-3-4-3; purifying the subfraction Fr.M6-1-3-4-2 by preparative high performance liquid chromatography, eluting with methanol-water mixed solvent with volume ratio of 50:50, collecting chromatographic peaks with retention time of 33.1min, 41.5min and 45.0min respectively for the chromatographic column YMC-Pack ODS-A and flow rate of 3 m/min, and obtaining subfraction Fr.M6-1-3-4-2-1, subfraction Fr.M6-1-3-4-2-2 and subfraction Fr.M6-1-3-4-2-3; purifying the subfraction Fr.M6-1-3-4-2-1 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 35:65, collecting chromatographic peak with flow rate of 3 m/min and retention time of 11.9min to obtain compound dysosmse:Sup>A versipellis C;
Purifying the subfraction Fr.M6-1-3-4-2-3 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 30:70, collecting chromatographic peak with flow rate of 3 m/min and retention time of 28.5min, and collecting the compound dysosmse:Sup>A versipellis ketone B.
5. The method for preparing the flavonol compound dysosma versipellis ketone A-C, E-F according to claim 2, wherein 30kg of dysosma versipellis medicinal material is taken as a raw material, ethanol with the weight, volume and volume concentration of 95% is added into the raw material for each time, the heating reflux extraction is carried out for 3 times, the extraction temperature is 93 ℃, the extraction time is 1.2 hours each time, and 95% ethanol is recovered under reduced pressure, so as to obtain an extract of 95% ethanol; adding 4 times of ethanol with weight volume and volume concentration of 50% into the residue, reflux extracting for 1 time at 93 deg.C for 1.2 hr, and recovering 50% ethanol under reduced pressure to obtain extract 50% ethanol extract; mixing 95% ethanol extract and 50% ethanol extract, adding 8.0L absolute ethanol for dissolving, adding 8.0kg diatomite for adsorption, recovering solvent, sequentially adding 12L dichloromethane, ethyl acetate and methanol for eluting, and recovering solvent to obtain dichloromethane eluting part, ethyl acetate eluting part and methanol eluting part respectively; separating the methanol elution part by silica gel column chromatography, sequentially performing gradient elution by using a dichloromethane-methanol mixed solvent system with volume ratios of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:30, 100:50 and 0:100, wherein each gradient uses 45L eluent with the flow rate of 60mL/min and each 7.5L is a flow, collecting 54 flow parts, detecting and analyzing each flow part by silica gel thin layer chromatography, and using GF 254 The thin layer plate is heated for 3min at 105 ℃ by taking petroleum ether-acetone with the volume ratio of 4:5 and dichloromethane-methanol with the volume ratio of 5:2 as developing agents, respectively, and according to the detection result of thin layer chromatography, 1-12 parts, 13-24 parts, 25-30 parts, 31-36 parts, 37-38 parts, 39-42 parts, 43-44 parts, 45-48 parts and 49-54 parts are combined to obtain a component Fr.M1, a component Fr.M2, a component Fr.M3, a component Fr.M4, a component Fr.M5, a component Fr.M6, a component Fr.M7, a component Fr.M8 and a component Fr.M9;
component Fr.M4 via sepSeparating by hadex LH-20 gel column chromatography, eluting with 7.5L methanol at a flow rate of 7.5mL/min, collecting 50 fractions each 150mL as one fraction, and respectively combining 1-25 fractions, 26-44 fractions and 45-50 fractions by silica gel thin layer chromatography detection analysis to obtain a subfraction Fr.M4-1, a subfraction Fr.M4-2 and a subfraction Fr.M4-3; the subfraction Fr.M4-1 was separated by ODS column chromatography with MeOH-H in a volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out on the O mixed solvent system, each gradient is 3.75L, each gradient is a subcomponent, and subcomponent Fr.M4-1-1, subcomponent Fr.M4-1-2, subcomponent Fr.M4-1-3, subcomponent Fr.M4-1-4, subcomponent Fr.M4-1-5 and subcomponent Fr.M4-1-6 are obtained; separating the subfractions Fr.M4-1-2 by sephadex LH-20 gel column chromatography, eluting with 750mL of methanol, taking each 7.5mL as a fraction, collecting 100 fractions, respectively combining 1-17, 18-41, 42-59, 60-77, 78-92 and 93-100 by silica gel thin layer chromatography detection analysis, and collecting the subfractions Fr.M4-1-2-1, fr.M4-1-2-3, fr.M4-1-2-4, fr.M4-1-2-5 and Fr.M4-1-2-6; purifying the subfraction M4-1-2-3 by preparative high performance liquid chromatography, eluting with se:Sup>A mixed solvent of methanol and water with se:Sup>A volume ratio of 52:48, collecting chromatographic peaks with retention time of 7.9min, 8.3min, 10.8min, 12.8min, 16.4min and 25.1min respectively for the chromatographic column YMC-Pack ODS-A, the flow rate of 3 m/min, and obtaining subfraction Fr.M4-1-2-3-1, subfraction Fr.M4-1-2-3-2, subfraction Fr.M4-1-2-3-3, subfraction Fr.M4-1-2-3-4, subfraction Fr.M4-1-2-3-5 and subfraction Fr.M4-1-2-3-6; purifying the subfraction Fr.M4-1-2-3-3 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peaks with retention time of 17.5min respectively, wherein the chromatographic column is YMC-Pack ODS-A, and the flow rate is 3 m/min, to obtain dysosmse:Sup>A versipellis E;
Purifying the subfraction Fr.M4-1-2-3-4 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 27:73, collecting chromatographic peak with flow rate of 3 m/min and retention time of 30.7min to obtain dysosmse:Sup>A versipellis ketone F;
separating the component Fr.M5 by sephadex LH-20 column chromatographyEluting with 3.75L of methanol at a flow rate of 7.5mL/min, collecting 25 fractions per 150mL, and performing silica gel thin layer chromatography detection analysis on each fraction, wherein 1-3, 4-5, 6-7, 8-10, 11-13, 14-17, 19-21 and 22-25 of fractions are combined to obtain a subfraction Fr.M5-1, a subfraction Fr.M5-2, a subfraction Fr.M5-3, a subfraction Fr.M5-4, a subfraction Fr.M5-5, a subfraction Fr.M5-6, a subfraction Fr.M5-7 and a subfraction Fr.M5-8; the subfractions Fr.M5-1 to Fr.M5-5 are combined, separated by MCI column chromatography and separated by MeOH-H with volume ratio of 0:100, 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, each gradient elution solvent is 1.5L, the flow rate is 3.7mL/min, each 375mL is a fraction, 28 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-5 fractions, 6-15 fractions, 16-23 fractions and 17-28 fractions are respectively combined to obtain a subfraction Fr.M5-1, a subfraction Fr.M5-1-2, a subfraction Fr.M5-1-3 and a subfraction Fr.M5-1-4; the subfractions Fr.M5-1-3 were separated by column chromatography on silica gel with CHCl in a volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 7:1, 3:1 3 Gradient elution of MeOH mixed solvent systems, wherein each gradient elution solvent is 75mL, the flow rate is 4.0mL/min, each 7.5mL is one fraction, 80 fractions are collected, each fraction is subjected to detection analysis by silica gel thin layer chromatography, and 1-10 fractions, 11-20 fractions, 21-30 fractions, 31-40 fractions, 41-46 fractions, 47-51 fractions, 52-60 fractions, 61-70 fractions and 71-80 fractions are respectively combined to obtain subfractions Fr.M5-1-3-1, subfractions Fr.M5-1-3-2, subfractions Fr.M5-1-3-3, subfractions Fr.M5-1-3-4, subfractions Fr.M5-1-3-5, subfractions Fr.M5-1-3-6, subfractions Fr.M5-1-3-7, subfractions Fr.M5-1-3-8 and subfractions Fr.M5-1-3-9; purifying the subfraction Fr.M5-1-3-5 by preparative high performance liquid chromatography, eluting with methanol-water mixed solvent with volume ratio of 55:45, collecting chromatographic peak with retention time of 34.9min at flow rate of 3 m/min with YMC-Pack ODS-A column to obtain dysosmse:Sup>A versipellis ketone A;
separating the component Fr.M6 by sephadex LH-20 column chromatography, eluting with 22.5L methanol at a flow rate of 7.5mL/min, collecting 30 fractions each 0.75L as one fraction, and respectively combining fractions 1-18 and 19-30 by silica gel thin layer chromatography detection analysis to obtain sub-componentsFr.m6-1 and fr.m6-2; the subfraction Fr.M6-1 is separated by ODS column chromatography with MeOH-H in volume ratio of 10:90, 30:70, 50:50, 70:30, 90:10, 100:0 2 Gradient elution is carried out by an O mixed solvent system, each gradient elution solvent is 3.75L, the flow rate is 5.0mL/min, each 0.75L is one fraction, 30 fractions are collected, each fraction is detected and analyzed by silica gel thin layer chromatography, and 1-5 fractions, 6-10 fractions, 11-20 fractions and 21-30 fractions are combined to obtain a subfraction Fr.M6-1-1, a subfraction Fr.M6-1-2, a subfraction Fr.M6-1-3 and a subfraction M6-1-4; separating the subfractions Fr.M6-1-3 by silica gel column chromatography, and performing gradient elution by using a methylene dichloride-methanol mixed solvent system with the volume ratio of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10 and 100:30, wherein each gradient elution solvent is 185L, the flow rate is 1.5mL/min, and each elution gradient is a flow part, so as to obtain subfractions Fr.M6-1, subfractions Fr.M6-1-3-2, subfractions Fr.M6-1-3-3, subfractions Fr.M6-1-3-4, subfractions Fr.M6-1-3-5, subfractions Fr.M6-1-3-6 and subfractions Fr.M6-1-3-7; separating the subfractions Fr.M6-1-3-4 by sephadex LH-20 gel column chromatography, eluting with 175mL of methanol, wherein the flow rate is 1.5mL/min, each 7mL is a fraction, collecting 25 fractions, and respectively combining the fractions 1-7, 8-16 and 17-25 by silica gel thin layer chromatography detection analysis to obtain subfractions Fr.M6-1-3-4-1, subfractions Fr.M6-1-3-4-2 and subfractions Fr.M6-1-3-4-3; purifying the subfraction Fr.M6-1-3-4-2 by preparative high performance liquid chromatography, eluting with methanol-water mixed solvent with volume ratio of 50:50, collecting chromatographic peaks with retention time of 33.1min, 41.5min and 45.0min respectively for the chromatographic column YMC-Pack ODS-A and flow rate of 3 m/min, and obtaining subfraction Fr.M6-1-3-4-2-1, subfraction Fr.M6-1-3-4-2-2 and subfraction Fr.M6-1-3-4-2-3; purifying the subfraction Fr.M6-1-3-4-2-1 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 35:65, collecting chromatographic peak with flow rate of 3 m/min and retention time of 11.9min to obtain compound dysosmse:Sup>A versipellis C;
Purifying the subfraction Fr.M6-1-3-4-2-3 by preparative high performance liquid chromatography, eluting with acetonitrile-water mixed solvent with volume ratio of 30:70, collecting chromatographic peak with flow rate of 3 m/min and retention time of 28.5min, and collecting the compound dysosmse:Sup>A versipellis ketone B.
6. Use of the flavonol compound dysosma versipellis a-C, E-F as claimed in claim 1 for preparing food or beverage antioxidants.
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| CN104666383A (en) * | 2014-12-30 | 2015-06-03 | 铜仁学院 | Technology for microwave-assisted extraction of dysosma versipellis flavonoids compound |
| CN106749303A (en) * | 2017-01-19 | 2017-05-31 | 浙江大学 | A kind of method for preparing podophyllotoxin and Dysosma versipellis biflavone and the like |
| CN107446009A (en) * | 2017-09-05 | 2017-12-08 | 广西中医药大学 | The O β D glucuronic acid methyl esters of chrysoeriol 7 and its extracting method and purposes |
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