CN111303104B - Preparation method and application of 7-hydroxy-8-methoxy coumarin standard substance - Google Patents
Preparation method and application of 7-hydroxy-8-methoxy coumarin 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 66
- RQSKEMWBCJHQMX-UHFFFAOYSA-N isoscopoletin Natural products COc1cc2OC(=O)CCc2cc1O RQSKEMWBCJHQMX-UHFFFAOYSA-N 0.000 title claims abstract description 65
- 239000000126 substance Substances 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000004440 column chromatography Methods 0.000 claims abstract description 20
- 238000000605 extraction Methods 0.000 claims abstract description 19
- 235000013305 food Nutrition 0.000 claims abstract description 3
- 238000004519 manufacturing process Methods 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 42
- 229910002027 silica gel Inorganic materials 0.000 claims description 42
- 239000002904 solvent Substances 0.000 claims description 17
- 238000010298 pulverizing process Methods 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 15
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical compound CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 238000001704 evaporation 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
- 239000000284 extract Substances 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 238000002156 mixing Methods 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 7
- 239000000843 powder Substances 0.000 claims description 7
- 238000002791 soaking Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 239000002994 raw material Substances 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 4
- 239000012043 crude product Substances 0.000 claims description 3
- 238000007689 inspection Methods 0.000 claims description 3
- 238000010829 isocratic elution Methods 0.000 claims description 2
- 230000011218 segmentation Effects 0.000 claims description 2
- 238000009826 distribution Methods 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 5
- 239000012846 chemical reference substance Substances 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 34
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- 239000000243 solution Substances 0.000 description 19
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 15
- 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
- 239000007788 liquid Substances 0.000 description 4
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 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
- 241000934856 Daphne Species 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 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
- 229960000956 coumarin Drugs 0.000 description 2
- 235000001671 coumarin Nutrition 0.000 description 2
- YBGKGTOOPNQOKH-UHFFFAOYSA-N daphnetin Natural products OC1=CC=CC2=C1OC(=O)C=C2O YBGKGTOOPNQOKH-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000002024 ethyl acetate extract Substances 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 241000123650 Botrytis cinerea Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241001163443 Daphne giraldii Species 0.000 description 1
- 241000711549 Hepacivirus C Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 244000168667 Pholiota nameko Species 0.000 description 1
- 235000014528 Pholiota nameko Nutrition 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
- 240000009022 Smilax rotundifolia Species 0.000 description 1
- 235000003205 Smilax rotundifolia Nutrition 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
- 229940124599 anti-inflammatory drug Drugs 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
- -1 coumarin compound Chemical class 0.000 description 1
- 150000004775 coumarins 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
- 239000003480 eluent Substances 0.000 description 1
- 239000000469 ethanolic extract Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 210000003918 fraction a Anatomy 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000005764 inhibitory process Effects 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
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 238000005325 percolation 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
- 239000012488 sample solution Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
Classifications
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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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention aims to provide a preparation method of a 7-hydroxy-8-methoxy coumarin standard substance, which is obtained by sequentially percolating and extracting with ethanol solution, segmenting by n-hexane extraction, separating by column chromatography and purifying. The pure 7-hydroxy-8-methoxycoumarin product 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 simple operation, high extraction efficiency, and extraction purity up to more than 99%, and the purity 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 in particular relates to a preparation method and application of a 7-hydroxy-8-methoxycoumarin standard substance.
Background
7-hydroxy-8-methoxycoumarin, english name of which is hydrongetin, CAS of 485-90-5, molecular formula of which is C 10 H 8 O 4 The molecular weight is 192.1, and the structural formula is as follows:
the extraction and preparation method of 7-hydroxy-8-methoxycoumarin is little disclosed, wherein the following method for extracting 7-hydroxy-8-methoxycoumarin is disclosed in China patent with application number of 201110384488.9 and with the name of coumarin compound application and method for extracting coumarin from daphne: collecting whole plant of daphne of daphnaceae, pulverizing, reflux extracting with 80-95% ethanol under heating, concentrating the extractive solution to obtain ethanol extract, dispersing with water, extracting with ethyl acetate, concentrating to obtain ethyl acetate extract, subjecting to silica gel column chromatography, gradient eluting with chloroform-methanol at volume ratio of 50:1, and eluting with chloroform: a gradient of 25:1 methanol volume ratio gives a fraction a, chloroform: a gradient of 10:1 methanol volume ratio gave a B fraction, chloroform: the method comprises the steps of obtaining a C fraction by a gradient of 8:1 of methanol volume ratio, separating three fractions by ODS column chromatography, eluting a A fraction by methanol-water of which the volume ratio is 6:4, eluting a B fraction by methanol-water of which the volume ratio is 1:9, eluting a C fraction by methanol-water of which the volume ratio is 1:9, and recrystallizing an eluent by methanol, wherein the A fraction is daphnetin, the B fraction is 7-hydroxy-8-methoxycoumarin, and the C fraction is daphnetin. The application also discloses that the inhibition rate of the 7-hydroxy-8-methoxycoumarin to the hepatitis C virus protease NS3/4A is 72.5% when the concentration of the 7-hydroxy-8-methoxycoumarin is 100 mg/mL.
The application number 200610030920.3 and the application of coumarin compounds in preparing anti-inflammatory and analgesic drugs disclose the following method for preparing 7-hydroxy-8-methoxycoumarin: 10Kg of root bark and stem bark of daphne giraldii herb is taken, crushed and then is subjected to percolation extraction by using 95% ethanol to obtain 50L of extract, the extract is concentrated and then is sequentially extracted by petroleum ether, chloroform, ethyl acetate and n-butanol, and the extract is concentrated into extractum. And performing systematic chemical component separation and identification on each extraction part by means of silica gel column chromatography, polyamide chromatography, sephadex LH-20, preparative HPLC and the like. As a result, 15 compounds were isolated at the ethyl acetate position, and structural identification was carried out, wherein 1 white powder was 7-hydroxy-8-methoxycoumarin. The application also discloses that the 7-hydroxy-8-methoxy coumarin has better anti-inflammatory and analgesic effects.
The purity of the 7-hydroxy-8-methoxycoumarin obtained by the method is difficult to meet the requirements of traditional Chinese medicine chemical reference substances. Through examination, the method for extracting the 7-hydroxy-8-methoxy coumarin with high purity as a standard substance is not disclosed.
Disclosure of Invention
The invention aims to solve the technical problems that: aiming at the defects existing in the prior art, the preparation method of the 7-hydroxy-8-methoxy coumarin standard substance is provided.
The invention solves the technical problems with the following technical proposal:
the preparation method of the 7-hydroxy-8-methoxy coumarin standard substance comprises the following specific steps:
(1) Percolating and extracting: pulverizing radix seu herba Heterophyllae into coarse powder, placing into a percolating cylinder, soaking in 6-12 times of 50-95% ethanol solution for 12-24 hr, percolating, collecting 10-20 times of percolate of the raw materials, and concentrating the percolate into soft extract;
(2) Extraction segmentation: adding 0-8% ethanol water solution 2-5 times of the thick paste to obtain suspension, adding n-hexane 1-2 times of the suspension, extracting for 3-5 times, mixing n-hexane solutions, recovering solvent, and concentrating to obtain thick paste;
(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 in a water bath, and crushing to obtain a mixed sample A; subjecting the mixed sample A to a silica gel chromatographic column, wherein the amount of the silica gel column is 30-100 times that of the mixed sample A, performing gradient elution with n-hexane-ethyl acetate, collecting fractions, performing thin layer chromatography for inspection, mixing fractions with 7-hydroxy-8-methoxycoumarin component, recovering solvent, and concentrating to obtain 7-hydroxy-8-methoxycoumarin crude product;
(4) Purifying: adding 2-4 times of column chromatography silica gel into the crude 7-hydroxy-8-methoxycoumarin product again, stirring uniformly, evaporating to dryness in water bath, and pulverizing to obtain a mixed sample B; subjecting the mixed sample B to a silica gel chromatographic column, eluting with 30-100 times of the mixed sample B with n-hexane-ethyl acetate, collecting the fraction, inspecting with thin layer chromatography, collecting the fraction containing 7-hydroxy-8-methoxycoumarin component, recovering solvent, and concentrating to obtain 7-hydroxy-8-methoxycoumarin pure product;
the times of the invention are calculated by mass.
The following technical scheme is used 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 collected fractions were collected in an amount of 1/4 to 1/2 of the column volume of the silica gel column divided in one fraction.
The isocratic elution mobile phase composition ratio of the n-hexane-ethyl acetate in the step (4) is 90:10-70:30; the collected fractions were collected in an amount of 1/4 to 1/2 of the column volume of the silica gel column divided in one fraction.
The invention also claims the application of the 7-hydroxy-8-methoxycoumarin pure product prepared by the method as a standard substance in the detection of 7-hydroxy-8-methoxycoumarin components of related products in the field of medicine or food production and circulation.
The extraction method has simple operation, high extraction efficiency, and extraction purity up to more than 99%, and the purity 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 of 7-hydroxy-8-methoxycoumarin.
Fig. 2 is an HPLC chromatogram of a test sample of glabrous greenbrier rhizome in the example, 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 used 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 pholiota nameko used in the examples of the present invention was collected from the county of gold, the Guangxi, and identified by the main pharmacist of the Guangxi traditional Chinese medicine institute Lu Wenjie as whole grass of the plant of the physcotoma pubescentis of the family hydrangeae.
1. Extraction preparation example
1. High purity 7-hydroxy-8-methoxycoumarin extraction example
Pulverizing radix seu herba Heterophyllae into coarse powder, placing into a percolating cylinder, soaking in 10 times of 90% ethanol solution for 16 hr, percolating, collecting 10 times of percolate of the raw materials, and concentrating into soft extract. Adding 5% ethanol water solution 2 times of the thick paste to obtain suspension, adding n-hexane 2 times of the suspension, extracting for 3 times, mixing n-hexane solutions, recovering solvent, and concentrating to obtain thick paste. Adding 3 times of column chromatography silica gel, stirring, evaporating to dryness in water bath, and pulverizing to obtain mixed sample A; the mixed sample A is subjected to a silica gel chromatographic column, the silica gel of the column chromatography is 50 times of the mixed sample A, the mixed sample A is subjected to gradient elution by n-hexane-ethyl acetate of 100:0- & gt 95:5- & gt 90:10- & gt 80:20- & gt 70:30- & gt 50:50, fractions are collected according to the 1/4-1/2 of the volume of the silica gel chromatographic column, the fractions are inspected by adopting thin layer chromatography, the fractions of 7-hydroxy-8-methoxycoumarin components can be detected, the solvent is recovered, and the mixture is concentrated, so that a crude 7-hydroxy-8-methoxycoumarin product is obtained. Adding 3 times of column chromatography silica gel again, stirring, evaporating to dryness in water bath, and pulverizing to obtain mixed sample B; and (3) allowing the mixed sample B to pass through a silica gel chromatographic column, eluting the mixed sample B with 100 times of the amount of the silica gel by using 70:30 isocratic n-hexane-ethyl acetate, collecting fractions according to 1/4-1/2 of the volume of the silica gel chromatographic column as one fraction, inspecting by using 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.
2. High-purity 7-hydroxy-8-methoxycoumarin extraction example II
Pulverizing radix seu herba Heterophyllae, soaking in 12 times of 95% ethanol solution for 12 hr, percolating, collecting 12 times of percolate, and concentrating into soft extract. Adding 5 times of water to obtain suspension, extracting with 2 times of n-hexane 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; the mixed sample A is subjected to a silica gel chromatographic column, the silica gel of the column chromatography is 100 times of the mixed sample A, the mixed sample A is subjected to gradient elution by n-hexane-ethyl acetate of 100:0- & gt 95:5- & gt 90:10- & gt 80:20- & gt 70:30- & gt 50:50, fractions are collected according to the 1/4-1/2 of the volume of the silica gel chromatographic column, the fractions are inspected by adopting thin layer chromatography, the fractions of 7-hydroxy-8-methoxycoumarin components can be detected, the solvent is recovered, and the mixture is concentrated to obtain a crude 7-hydroxy-8-methoxycoumarin product. Adding 3 times of column chromatography silica gel again, stirring, evaporating to dryness in water bath, and pulverizing to obtain mixed sample B; and (3) allowing the mixed sample B to pass through a silica gel chromatographic column, eluting the mixed sample B with 50 times of the amount of the silica gel by using n-hexane-ethyl acetate at a ratio of 80:20, collecting fractions according to 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 a solvent, and concentrating to obtain a pure 7-hydroxy-8-methoxycoumarin product.
3. High purity 7-hydroxy-8-methoxycoumarin extraction example three
Pulverizing radix seu herba Heterophyllae into coarse powder, placing in a percolating cylinder, soaking in 10 times of 85% ethanol solution for 15 hr, percolating, collecting 10 times of percolate of the raw materials, and concentrating into soft extract. Adding 3% ethanol water solution 3 times of the thick paste to obtain suspension, adding n-hexane 2 times of the suspension, extracting for 4 times, mixing n-hexane solutions, recovering solvent, and concentrating to obtain thick paste. Adding 4 times of column chromatography silica gel, stirring, evaporating to dryness in water bath, and pulverizing to obtain mixed sample A; the mixed sample A is subjected to a silica gel chromatographic column, the silica gel of the column chromatography is 50 times of the mixed sample A, the mixed sample A is subjected to gradient elution by n-hexane-ethyl acetate of 100:0- & gt 95:5- & gt 90:10- & gt 80:20- & gt 70:30- & gt 50:50, fractions are collected according to the 1/4-1/2 of the volume of the silica gel chromatographic column, the fractions are inspected by adopting thin layer chromatography, the fractions of 7-hydroxy-8-methoxycoumarin components can be detected, the solvent is recovered, and the mixture is concentrated, so that a crude 7-hydroxy-8-methoxycoumarin product is obtained. Adding 4 times of column chromatography silica gel again, stirring, evaporating to dryness in water bath, and pulverizing to obtain mixed sample B; and (3) allowing the mixed sample B to pass through a silica gel chromatographic column, eluting the mixed sample B with 50 times of the amount of the silica gel by using n-hexane-ethyl acetate at 90:10 isocratic, collecting fractions according to 1/4-1/2 of the volume of the silica gel chromatographic column as one fraction, inspecting by using 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.
4. Comparative example was extracted
Pulverizing radix seu herba Heterophyllae into coarse powder, placing in a percolating cylinder, soaking in 10 times of 85% ethanol solution for 15 hr, percolating, collecting 10 times of percolate of the raw materials, and concentrating into soft extract. Adding 3% ethanol water solution 3 times of the thick paste to obtain suspension, adding ethyl acetate 2 times of the suspension, extracting for 4 times, mixing ethyl acetate extracts, recovering solvent, and concentrating to obtain thick paste. Adding 4 times of column chromatography silica gel, stirring, evaporating to dryness in water bath, and pulverizing to obtain mixed sample A; the mixed sample A is passed through a silica gel chromatographic column, the silica gel of the column chromatography is 50 times of the mixed sample A, the mixed sample A is eluted by 10:1 chloroform-methanol, fractions are collected according to the 1/4-1/2 of the volume of the silica gel chromatographic column, the thin layer chromatography is adopted for inspection, the fractions of the 7-hydroxy-8-methoxycoumarin component can be detected are combined, the solvent is recovered, and the concentration is carried out, so that the crude 7-hydroxy-8-methoxycoumarin product is obtained. Separating the crude product by ODS column chromatography, eluting with methanol-water at volume ratio of 1:9, and recrystallizing the eluate with methanol to obtain pure 7-hydroxy-8-methoxycoumarin.
2. Structure confirmation and purity measurement result
The spectrum resolution 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 ]] + 。 1 H-NMR(800MHz，CDCl 3 )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，-iCH 3 )； 13 C-NMR(200MHz，CDCl 3 )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-OCH 3 ). And thus identified as 7-hydroxy-8-methoxycoumarin.
The purity of the 7-hydroxy-8-methoxycoumarin obtained in the four extraction examples was 99.5%, 99.0%, 99.2% and 93.6%, respectively, as determined by purity.
3. Quality control for measuring content of 7-hydroxy-8-methoxy coumarin by HPLC method of Botrytis cinerea
1. Chromatographic conditions
Liquid chromatographic column (chromatograph) with octadecylsilane chemically bonded silica as filler by reversed phase high performance liquid chromatographyColumn:c18 5 μm (4.6X1250 mm)), methanol-0.1% phosphoric acid (22:78) as mobile phase; the flow rate is 1ml/min; column temperature: 25 ℃; detection wavelength: 325nm; and calculating the content of the sample by adopting an area normalization method.
2. Preparation of control solution
Taking 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 prepare a solution containing 40 mug of 7-hydroxy-8-methoxycoumarin per 1 ml.
3. Preparation of test solutions
Taking about 0.5g of star-crown vine powder (sieving with a No. 6 sieve), precisely weighing, placing into a conical bottle with a plug, precisely adding 50ml of methanol, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, and collecting the subsequent filtrate.
4. Measurement
Respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
5. The liquid chromatogram is shown in fig. 1 and 2. Fig. 1 is an HPLC chromatogram of a control, tr= 23.277 as a chromatographic peak of 7-hydroxy-8-methoxycoumarin. Fig. 2 is an HPLC chromatogram of a test sample, with tr= 23.383 being the chromatographic peak of 7-hydroxy-8-methoxycoumarin.
Claims (4)
1. The preparation method of the 7-hydroxy-8-methoxy coumarin standard substance is characterized by comprising the following specific steps of:
(1) Percolating and extracting: pulverizing radix seu herba Heterophyllae into coarse powder, placing into a percolating cylinder, soaking in 6-12 times of 85-95% ethanol solution for 12-24 hr, percolating, collecting 10-20 times of percolate of the raw materials, and concentrating the percolate into soft extract;
(2) Extraction segmentation: adding 0-8% ethanol water solution 2-5 times of the thick paste to obtain suspension, adding n-hexane 1-2 times of the suspension, extracting for 3-5 times, mixing n-hexane solutions, recovering solvent, and concentrating to obtain thick paste;
(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 in a water bath, and crushing to obtain a mixed sample A; subjecting the mixed sample A to a silica gel chromatographic column, wherein the amount of the silica gel column is 30-100 times that of the mixed sample A, performing gradient elution with n-hexane-ethyl acetate, collecting fractions, performing thin layer chromatography for inspection, mixing fractions with 7-hydroxy-8-methoxycoumarin component, recovering solvent, and concentrating to obtain 7-hydroxy-8-methoxycoumarin crude product;
(4) Purifying: adding 2-4 times of column chromatography silica gel into the crude 7-hydroxy-8-methoxycoumarin product again, stirring uniformly, evaporating to dryness in water bath, and pulverizing to obtain a mixed sample B; subjecting the mixed sample B to a silica gel chromatographic column, eluting with 30-100 times of the mixed sample B with n-hexane-ethyl acetate, collecting the fraction, inspecting with thin layer chromatography, collecting the fraction containing 7-hydroxy-8-methoxycoumarin component, recovering solvent, and concentrating to obtain 7-hydroxy-8-methoxycoumarin pure product;
the times of the invention are calculated by mass;
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 isocratic elution mobile phase composition ratio of the n-hexane-ethyl acetate in the step (4) is 90:10-70:30.
2. The method for preparing 7-hydroxy-8-methoxycoumarin standard substance according to claim 1, wherein the collected fraction in step (3) is collected in an amount of 1/4 to 1/2 of the column volume of the silica gel column divided into one fraction.
3. The method for preparing 7-hydroxy-8-methoxycoumarin standard substance according to claim 1, wherein the collected fraction in step (4) is collected in an amount of 1/4 to 1/2 of the column volume of the silica gel column divided into one fraction.
4. Use of the pure 7-hydroxy-8-methoxycoumarin product obtained in any one of claims 1-3 as a standard substance for detecting 7-hydroxy-8-methoxycoumarin components in related products in the field of pharmaceutical or food production and distribution.
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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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| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 瑶族药消瘤藤的化学成分分析;陆国寿等;《中国实验方剂学杂志》;20170731(第14期);第79页第2节第1、3段 * |
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