CN111303104A - Preparation method and application of 7-hydroxy-8-methoxycoumarin standard substance - Google Patents
Preparation method and application of 7-hydroxy-8-methoxycoumarin standard substance Download PDFInfo
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- HAQWEMHXSIRYBE-UHFFFAOYSA-N 7-hydroxy-8-methoxycoumarin Chemical compound C1=CC(=O)OC2=C1C=CC(O)=C2OC HAQWEMHXSIRYBE-UHFFFAOYSA-N 0.000 title claims abstract description 67
- RQSKEMWBCJHQMX-UHFFFAOYSA-N isoscopoletin Natural products COc1cc2OC(=O)CCc2cc1O RQSKEMWBCJHQMX-UHFFFAOYSA-N 0.000 title claims abstract description 66
- 239000000126 substance Substances 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 238000004440 column chromatography Methods 0.000 claims abstract description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 23
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000000926 separation method Methods 0.000 claims abstract description 4
- 235000013305 food Nutrition 0.000 claims abstract description 3
- 238000004519 manufacturing process Methods 0.000 claims abstract description 3
- 238000005325 percolation Methods 0.000 claims abstract description 3
- 238000000746 purification Methods 0.000 claims abstract description 3
- 230000011218 segmentation Effects 0.000 claims abstract description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 44
- 239000000741 silica gel Substances 0.000 claims description 44
- 229910002027 silica gel Inorganic materials 0.000 claims description 44
- 239000000243 solution Substances 0.000 claims description 24
- 239000000284 extract Substances 0.000 claims description 20
- 239000002904 solvent Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical compound CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 claims description 11
- 238000001704 evaporation Methods 0.000 claims description 11
- 238000010298 pulverizing process Methods 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 11
- 239000000725 suspension Substances 0.000 claims description 11
- 238000004809 thin layer chromatography Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 10
- 239000012043 crude product Substances 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 7
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 5
- 238000010829 isocratic elution Methods 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims 1
- 238000009826 distribution Methods 0.000 claims 1
- 239000012264 purified product Substances 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 4
- 239000012846 chemical reference substance Substances 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 30
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 18
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- ATEFPOUAMCWAQS-UHFFFAOYSA-N 7,8-dihydroxycoumarin Chemical compound C1=CC(=O)OC2=C(O)C(O)=CC=C21 ATEFPOUAMCWAQS-UHFFFAOYSA-N 0.000 description 2
- 241000284156 Clerodendrum quadriloculare Species 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 2
- 150000004775 coumarins Chemical class 0.000 description 2
- YBGKGTOOPNQOKH-UHFFFAOYSA-N daphnetin Natural products OC1=CC=CC2=C1OC(=O)C=C2O YBGKGTOOPNQOKH-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 229940126680 traditional chinese medicines Drugs 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 241000934856 Daphne Species 0.000 description 1
- 241001163443 Daphne giraldii Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000711549 Hepacivirus C Species 0.000 description 1
- 241001092080 Hydrangea Species 0.000 description 1
- 235000014486 Hydrangea macrophylla Nutrition 0.000 description 1
- CJHJQWPAFJADLM-UHFFFAOYSA-N OC1=CC=C2C=CC(OC2=C1OC)=O.OC1=CC=C2C=CC(OC2=C1OC)=O Chemical compound OC1=CC=C2C=CC(OC2=C1OC)=O.OC1=CC=C2C=CC(OC2=C1OC)=O CJHJQWPAFJADLM-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241001534930 Thymelaeaceae Species 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940035676 analgesics Drugs 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 230000001760 anti-analgesic effect Effects 0.000 description 1
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229960000956 coumarin Drugs 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- -1 coumarin compound Chemical class 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002451 electron ionisation mass spectrometry Methods 0.000 description 1
- 239000000469 ethanolic extract Substances 0.000 description 1
- 239000002024 ethyl acetate extract Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 210000002196 fr. b Anatomy 0.000 description 1
- 210000003918 fraction a Anatomy 0.000 description 1
- KIGCGZUAVODHMD-UHFFFAOYSA-N hydrangetin Natural products C1=CC(=O)OC2=C(O)C(OC)=CC=C21 KIGCGZUAVODHMD-UHFFFAOYSA-N 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 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 description 1
- 238000010606 normalization Methods 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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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
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
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- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention aims to provide a preparation method of a 7-hydroxy-8-methoxycoumarin standard substance, which is obtained by sequentially carrying out percolation extraction on an ethanol solution, n-hexane extraction segmentation, column chromatography separation and purification. The pure 7-hydroxy-8-methoxycoumarin obtained by the invention can be used as a standard substance for detecting the 7-hydroxy-8-methoxycoumarin component in related products in the field of medicine or food production and circulation. The extraction method has the advantages of simple operation, high extraction efficiency, high extraction purity of more than 99 percent and high purity of the product, and can meet the requirements of chemical reference substances.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to separation of natural traditional Chinese medicines, and particularly relates to a preparation method and application of a 7-hydroxy-8-methoxycoumarin standard substance.
Background
7-hydroxy-8-methoxycoumarin (English name: Hydrangetin, CAS: 485-90-5) with molecular formula of C10H8O4Molecular weight 192.1, structure thereofThe formula is as follows:
the extraction and preparation methods of 7-hydroxy-8-methoxycoumarin are rarely disclosed, wherein Chinese patents with application number of 201110384488.9 and name of application of coumarin compounds and extraction method of coumarin compounds from daphne disclose the following methods for extracting 7-hydroxy-8-methoxycoumarin: pulverizing whole plant of Thymelaeaceae, extracting with 80-95% ethanol under reflux, concentrating the extractive solution to obtain ethanol extract, dispersing with water, extracting with ethyl acetate, concentrating to obtain ethyl acetate extract, performing silica gel column chromatography, gradient eluting with chloroform-methanol at volume ratio of 50:1, eluting in chloroform: gradient of methanol at 25:1 volume ratio to obtain fraction a, chloroform: gradient of methanol at a volume ratio of 10:1 to obtain fraction B, chloroform: gradient with methanol volume ratio of 8:1 to obtain C fraction, separating three fractions by ODS column chromatography, eluting A fraction with methanol-water with volume ratio of 6:4, eluting B fraction with methanol-water with volume ratio of 1:9, eluting C fraction with methanol-water with volume ratio of 1:9, recrystallizing the eluates with methanol, respectively, obtaining daphnetin from A fraction, obtaining 7-hydroxy-8-methoxycoumarin from B fraction, and obtaining daphnetin from C fraction. The application also discloses that the inhibition rate of the 7-hydroxy-8-methoxycoumarin on the hepatitis C virus protease NS3/4A is 72.5% when the concentration is 100 mg/mL.
In addition, the application number of 200610030920.3, named as application of coumarin compound in preparing anti-inflammatory analgesic drugs, discloses the following method for preparing 7-hydroxy-8-methoxy coumarin: taking 10Kg of root bark and stem bark of daphne giraldii nitsche medicinal material, crushing, percolating and extracting with 95% ethanol to obtain 50L of extract, sequentially extracting with petroleum ether, chloroform, ethyl acetate and n-butanol after concentrating, and concentrating the extract into extract. And (3) separating and identifying chemical components of each extraction part by means of silica gel column chromatography, polyamide chromatography, Sephadex LH-20, preparative HPLC and the like. The results separated in ethyl acetate position to obtain 15 compounds, and the structure identification shows that 1 white powder is 7-hydroxy-8-methoxy coumarin. The application also discloses that the 7-hydroxy-8-methoxy coumarin has good anti-inflammatory and analgesic effects.
The purity of the 7-hydroxy-8-methoxy coumarin obtained by the method is difficult to meet the requirements of chemical reference substances of traditional Chinese medicines. According to the inspection, the method for extracting the 7-hydroxy-8-methoxycoumarin as the standard substance in high purity is not disclosed.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the defects in the prior art, the preparation method of the 7-hydroxy-8-methoxycoumarin standard substance is provided.
The invention solves the technical problems by the following technical scheme:
a preparation method of a 7-hydroxy-8-methoxycoumarin standard substance comprises the following specific steps:
(1) percolation extraction: pulverizing herba Erodii seu Geranii into coarse powder, placing in a percolating tube, adding 6-12 times of 50-95% ethanol solution, soaking for 12-24 hr, percolating, collecting 10-20 times of percolate, and concentrating the percolate into soft extract;
(2) extraction and segmentation: adding 0-8% ethanol water solution 2-5 times the amount of the soft extract to obtain suspension, adding n-hexane 1-2 times the amount of the suspension, extracting for 3-5 times, mixing n-hexane solutions, recovering solvent, and concentrating to obtain soft extract;
(3) and (3) column chromatography separation: adding 2-4 times of column chromatography silica gel into the thick paste obtained in the step (2), uniformly stirring, evaporating to dryness on a water bath, and crushing to obtain a mixed sample A; passing the mixed sample A through a silica gel chromatographic column, performing column chromatography on silica gel which is 30-100 times of the mixed sample A, performing gradient elution by using n-hexane-ethyl acetate, collecting fractions, inspecting by adopting thin-layer chromatography, combining the fractions in which the 7-hydroxy-8-methoxycoumarin component can be detected, recovering the solvent, and concentrating to obtain a crude product of the 7-hydroxy-8-methoxycoumarin;
(4) and (3) purification: adding the crude product of the 7-hydroxy-8-methoxycoumarin into 2-4 times of column chromatography silica gel again, stirring uniformly, evaporating to dryness on a water bath, and crushing to obtain a mixed sample B; passing the mixed sample B through a silica gel chromatographic column, performing column chromatography on silica gel which is 30-100 times of the mixed sample B, isocratic elution by n-hexane-ethyl acetate, collecting fractions, inspecting by adopting thin-layer chromatography, collecting fractions only containing 7-hydroxy-8-methoxycoumarin components, recovering the solvent, and concentrating to obtain a pure 7-hydroxy-8-methoxycoumarin product;
the times of the weight of the composition are calculated by mass.
The following technical scheme is taken as a preferable scheme of the technical scheme:
the volume percentage of the ethanol solution in the step (1) is 85-95%.
The volume percentage of the ethanol water solution in the step (2) is 3-5%.
The elution gradient of the n-hexane-ethyl acetate in the step (3) is 100:0 → 95:5 → 90:10 → 80:20 → 70:30 → 50: 50; the fractions were collected as 1/4-1/2 fractions of a silica gel column volume fraction.
The proportion of the flowing phase of the n-hexane-ethyl acetate for isocratic elution in the step (4) is 90:10-70: 30; the fractions were collected as 1/4-1/2 fractions of a silica gel column volume fraction.
The invention also claims the application of the pure 7-hydroxy-8-methoxycoumarin prepared by the method as a standard substance in the detection of the 7-hydroxy-8-methoxycoumarin component of related products in the field of medicine or food production and circulation.
The extraction method has the advantages of simple operation, high extraction efficiency, high extraction purity of more than 99 percent and high purity of the product, and can meet the requirements of chemical reference substances.
Drawings
Fig. 1 is an HPLC chromatogram of a 7-hydroxy-8-methoxycoumarin control in example, with tR-23.277 being the chromatographic peak for 7-hydroxy-8-methoxycoumarin.
Fig. 2 is an HPLC chromatogram of the sample of coronaria stellata in example, wherein tR-23.383 is a chromatographic peak of 7-hydroxy-8-methoxycoumarin.
Detailed Description
The inventive concepts of the present solution will be described below using terms commonly employed by those skilled in the art to convey the substance of their work to others skilled in the art. These inventive concepts may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
The coronaria stelleriana used in the embodiment of the invention is collected from Guangxi Jinxiu county, and is identified as the whole herb of the coronaria stelleriana Pileostegiae hand, Mazz of hydrangea of the Guangxi traditional Chinese medicine research institute by Luvingjie Master-ren pharmacist.
First, extraction preparation example
1. Extraction example of high purity 7-hydroxy-8-methoxy coumarin
Pulverizing herba Erodii seu Geranii into coarse powder, placing in a percolating cylinder, adding 10 times of 90% ethanol solution, soaking for 16 hr, percolating, collecting percolate 10 times of the raw materials, and concentrating the percolate to obtain soft extract. Adding 5% ethanol water solution 2 times the amount of the soft extract to obtain suspension, adding n-hexane 2 times the amount of the suspension, extracting for 3 times, mixing n-hexane solutions, recovering solvent, and concentrating to obtain soft extract. Adding 3 times of column chromatography silica gel, stirring, evaporating to dryness in water bath, and pulverizing to obtain mixed sample A; and (2) passing the mixed sample A through a silica gel chromatographic column, carrying out gradient elution by using silica gel of column chromatography which is 50 times of the mixed sample A, collecting fractions according to the proportion that one fraction is 1/4-1/2 of the volume of the silica gel chromatographic column, carrying out inspection by adopting thin-layer chromatography, combining fractions in which a 7-hydroxy-8-methoxycoumarin component can be detected, recovering a solvent, and concentrating to obtain a crude product of the 7-hydroxy-8-methoxycoumarin. Adding 3 times of column chromatography silica gel again, stirring uniformly, evaporating to dryness on a water bath, and crushing to obtain a mixed sample B; and (3) passing the mixed sample B through a silica gel chromatographic column, carrying out column chromatography on silica gel which is 100 times of the mixed sample B, eluting at a constant rate of n-hexane-ethyl acetate 70:30, collecting fractions according to the condition that one fraction is 1/4-1/2 of the volume of the silica gel chromatographic column, inspecting by adopting thin-layer chromatography, collecting fractions only containing 7-hydroxy-8-methoxycoumarin components, recovering the solvent, and concentrating to obtain the pure 7-hydroxy-8-methoxycoumarin.
2. Extraction example II of high purity 7-hydroxy-8-methoxycoumarin
Pulverizing herba Erodii seu Geranii into coarse powder, placing in a percolating cylinder, soaking in 12 times of 95% ethanol solution for 12 hr, percolating, collecting percolate 12 times of the raw materials, and concentrating the percolate to obtain soft extract. Adding 5 times of water to obtain suspension, adding 2 times of n-hexane, extracting for 5 times, mixing n-hexane solutions, recovering solvent, and concentrating to obtain soft extract. Adding 3 times of column chromatography silica gel, stirring, evaporating to dryness in water bath, and pulverizing to obtain mixed sample A; and (2) passing the mixed sample A through a silica gel chromatographic column, carrying out gradient elution by using silica gel of column chromatography which is 100 times of the mixed sample A, collecting fractions according to the proportion that one fraction is 1/4-1/2 of the volume of the silica gel chromatographic column, carrying out inspection by adopting thin-layer chromatography, combining fractions in which a 7-hydroxy-8-methoxycoumarin component can be detected, recovering a solvent, and concentrating to obtain a crude product of the 7-hydroxy-8-methoxycoumarin. Adding 3 times of column chromatography silica gel again, stirring uniformly, evaporating to dryness on a water bath, and crushing to obtain a mixed sample B; and (3) passing the mixed sample B through a silica gel chromatographic column, carrying out column chromatography on silica gel which is 50 times of the mixed sample B, eluting at a constant rate of 80:20 by using n-hexane-ethyl acetate, collecting fractions according to the condition that one fraction is 1/4-1/2 of the volume of the silica gel chromatographic column, inspecting by using thin-layer chromatography, collecting fractions only containing 7-hydroxy-8-methoxycoumarin components, recovering the solvent, and concentrating to obtain the pure 7-hydroxy-8-methoxycoumarin.
3. Extraction example III of highly pure 7-hydroxy-8-methoxycoumarin
Pulverizing herba Erodii seu Geranii into coarse powder, placing in a percolating cylinder, soaking in 10 times of 85% ethanol solution for 15 hr, percolating, collecting percolate 10 times of the raw materials, and concentrating the percolate to obtain soft extract. Adding 3% ethanol water solution 3 times the amount of the soft extract to obtain suspension, adding n-hexane 2 times the amount of the suspension, extracting for 4 times, mixing n-hexane solutions, recovering solvent, and concentrating to obtain soft extract. Adding 4 times of column chromatography silica gel, stirring, evaporating to dryness in water bath, and pulverizing to obtain mixed sample A; and (2) passing the mixed sample A through a silica gel chromatographic column, carrying out gradient elution by using silica gel of column chromatography which is 50 times of the mixed sample A, collecting fractions according to the proportion that one fraction is 1/4-1/2 of the volume of the silica gel chromatographic column, carrying out inspection by adopting thin-layer chromatography, combining fractions in which a 7-hydroxy-8-methoxycoumarin component can be detected, recovering a solvent, and concentrating to obtain a crude product of the 7-hydroxy-8-methoxycoumarin. Adding 4 times of column chromatography silica gel again, stirring uniformly, evaporating to dryness on a water bath, and crushing to obtain a mixed sample B; and (3) passing the mixed sample B through a silica gel chromatographic column, carrying out column chromatography on silica gel which is 50 times of the mixed sample B, eluting at an isocratic rate of n-hexane-ethyl acetate 90:10, collecting fractions according to the condition that one fraction is 1/4-1/2 of the volume of the silica gel chromatographic column, inspecting by adopting thin-layer chromatography, collecting fractions only containing 7-hydroxy-8-methoxycoumarin components, recovering the solvent, and concentrating to obtain the pure 7-hydroxy-8-methoxycoumarin.
4. Extraction comparative example
Pulverizing herba Erodii seu Geranii into coarse powder, placing in a percolating cylinder, soaking in 10 times of 85% ethanol solution for 15 hr, percolating, collecting percolate 10 times of the raw materials, and concentrating the percolate to obtain soft extract. Adding 3% ethanol water solution 3 times the amount of the soft extract to obtain suspension, adding ethyl acetate 2 times the amount of the suspension, extracting for 4 times, mixing ethyl acetate extractive solutions, recovering solvent, and concentrating to obtain soft extract. Adding 4 times of column chromatography silica gel, stirring, evaporating to dryness in water bath, and pulverizing to obtain mixed sample A; and (2) passing the mixed sample A through a silica gel chromatographic column, carrying out column chromatography on silica gel which is 50 times of the mixed sample A, eluting with 10:1 chloroform-methanol, collecting fractions according to the proportion that one fraction is 1/4-1/2 of the volume of the silica gel chromatographic column, inspecting by adopting thin-layer chromatography, combining the fractions which can detect the 7-hydroxy-8-methoxycoumarin component, recovering the solvent, and concentrating to obtain a crude product of the 7-hydroxy-8-methoxycoumarin. Separating the crude product by ODS column chromatography, eluting with methanol-water at a volume ratio of 1:9, and recrystallizing the eluate with methanol to obtain pure 7-hydroxy-8-methoxycoumarin.
II, structure confirmation and purity determination results
The data of the 7-hydroxy-8-methoxycoumarin obtained in the four extraction examples are as follows: colorless square crystals can be precipitated from ethyl acetate. EI-MS m/z: 192[ M ]]+。1H-NMR(800MHz，CDCl3)δ7.63(1H，d，J＝9.5Hz，H-4)，7.11(1H，d，J＝8.5Hz，H-5)，6.90(1H，d，J＝8.5Hz，H-6)，6.24(1H，d，J＝9.5Hz，H-3)，4.12(3H，s，-iCH3)；13C-NMR(200MHz，CDCl3)δ160.5(C-2)，152.2(C-7)，147.3(C-8a)，144.4(C-4)，133.8(C-8)，123.4(C-5)，113.4(C-4a)，112.8(C-3)，112.2(C-6)，61.9(8-OCH3). Therefore, it was identified as 7-hydroxy-8-methoxycoumarin (7-hydroxy-8-methoxycoumarin).
The purity of the 7-hydroxy-8-methoxy coumarin obtained in the four extraction examples is 99.5%, 99.0%, 99.2% and 93.6% respectively by purity determination.
Quality control method for determining 7-hydroxy-8-methoxy coumarin content by HPLC method of starburst coronaria
1. Chromatographic conditions
The measurement was performed by reverse phase high performance liquid chromatography using a liquid chromatography column (column:
c185 μm (4.6X 250mm)), with methanol-0.1% phosphoric acid (22:78) as mobile phase; the flow rate is 1 ml/min; column temperature: 25 ℃; detection wavelength: 325 nm; and calculating the content of the sample by adopting an area normalization method.
2. Preparation of control solutions
Taking the 7-hydroxy-8-methoxycoumarin obtained in the first extraction example as a standard reference substance, precisely weighing, placing in a brown measuring flask, and adding methanol to obtain a solution containing 40 μ g of 7-hydroxy-8-methoxycoumarin per 1 ml.
3. Preparation of test solution
Taking 0.5g of starburst coronary canopy vine powder (screened by a No. 6 sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of methanol, sealing the plug, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
4. Measurement of
Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
5. The liquid chromatogram is shown in FIGS. 1 and 2. Fig. 1 is an HPLC chromatogram of a control, and tR ═ 23.277 is the chromatographic peak of 7-hydroxy-8-methoxycoumarin. Fig. 2 is an HPLC chromatogram of the test sample, and tR ═ 23.383 is the chromatographic peak of 7-hydroxy-8-methoxycoumarin.
Claims (6)
1. A preparation method of a 7-hydroxy-8-methoxycoumarin standard substance is characterized by comprising the following specific steps:
(1) percolation extraction: pulverizing herba Erodii seu Geranii into coarse powder, placing in a percolating tube, adding 6-12 times of 50-95% ethanol solution, soaking for 12-24 hr, percolating, collecting 10-20 times of percolate, and concentrating the percolate into soft extract;
(2) extraction and segmentation: adding 0-8% ethanol water solution 2-5 times the amount of the soft extract to obtain suspension, adding n-hexane 1-2 times the amount of the suspension, extracting for 3-5 times, mixing n-hexane solutions, recovering solvent, and concentrating to obtain soft extract;
(3) and (3) column chromatography separation: adding 2-4 times of column chromatography silica gel into the thick paste obtained in the step (2), uniformly stirring, evaporating to dryness on a water bath, and crushing to obtain a mixed sample A; passing the mixed sample A through a silica gel chromatographic column, performing column chromatography on silica gel which is 30-100 times of the mixed sample A, performing gradient elution by using n-hexane-ethyl acetate, collecting fractions, inspecting by adopting thin-layer chromatography, combining the fractions in which the 7-hydroxy-8-methoxycoumarin component can be detected, recovering the solvent, and concentrating to obtain a crude product of the 7-hydroxy-8-methoxycoumarin;
(4) and (3) purification: adding the crude product of the 7-hydroxy-8-methoxycoumarin into 2-4 times of column chromatography silica gel again, stirring uniformly, evaporating to dryness on a water bath, and crushing to obtain a mixed sample B; passing the mixed sample B through a silica gel chromatographic column, performing column chromatography on silica gel which is 30-100 times of the mixed sample B, isocratic elution by n-hexane-ethyl acetate, collecting fractions, inspecting by adopting thin-layer chromatography, collecting fractions only containing 7-hydroxy-8-methoxycoumarin components, recovering the solvent, and concentrating to obtain a pure 7-hydroxy-8-methoxycoumarin product;
the times of the weight of the composition are calculated by mass.
2. The method for preparing 7-hydroxy-8-methoxycoumarin standard substance according to claim 1, wherein the ethanol solution of step (1) has a volume percentage of 85-95%.
3. The method for preparing 7-hydroxy-8-methoxycoumarin standard substance according to claim 1, wherein the ethanol aqueous solution in step (2) has a volume percentage of 3-5%.
4. The method for preparing 7-hydroxy-8-methoxycoumarin standard substance according to claim 1, wherein the elution gradient of n-hexane-ethyl acetate in step (3) is 100:0 → 95:5 → 90:10 → 80:20 → 70:30 → 50: 50; the fractions were collected as 1/4-1/2 fractions of a silica gel column volume fraction.
5. The method for preparing 7-hydroxy-8-methoxycoumarin standard substance according to claim 1, wherein the isocratic elution mobile phase composition ratio of n-hexane-ethyl acetate in the step (4) is 90:10-70: 30; the fractions were collected as 1/4-1/2 fractions of a silica gel column volume fraction.
6. The use of the purified product of 7-hydroxy-8-methoxycoumarin obtained according to any one of claims 1 to 5 as a standard substance for the detection of the 7-hydroxy-8-methoxycoumarin component in related products in the field of pharmaceutical or food production and distribution.
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| CN1923193A (en) * | 2006-09-07 | 2007-03-07 | 中国人民解放军第二军医大学 | Application of cumarin kind compound in preparation of antiphlogistic and analgetic medicine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1923193A (en) * | 2006-09-07 | 2007-03-07 | 中国人民解放军第二军医大学 | Application of cumarin kind compound in preparation of antiphlogistic and analgetic medicine |
| CN103027909A (en) * | 2011-11-28 | 2013-04-10 | 贵阳中医学院 | Application of coumarins compounds and method for extracting coumarins compounds from winter daphne |
| CN107064366A (en) * | 2017-04-20 | 2017-08-18 | 广西壮族自治区中医药研究院 | The content assaying method of Coumarins composition in Radix Pileostegiae tomentellae |
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