CN114634539B - Method for separating and preparing coumarin compound from anisodamine - Google Patents
Method for separating and preparing coumarin compound from anisodamine Download PDFInfo
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- CN114634539B CN114634539B CN202210454236.7A CN202210454236A CN114634539B CN 114634539 B CN114634539 B CN 114634539B CN 202210454236 A CN202210454236 A CN 202210454236A CN 114634539 B CN114634539 B CN 114634539B
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- WTQYWNWRJNXDEG-UHFFFAOYSA-N 6-Hydroxy-hyoscyamin Natural products CN1C(C2)CC(O)C1CC2OC(=O)C(CO)C1=CC=CC=C1 WTQYWNWRJNXDEG-UHFFFAOYSA-N 0.000 title claims abstract description 93
- WTQYWNWRJNXDEG-LEOABGAYSA-N anisodamine Chemical compound C1([C@@H](CO)C(=O)O[C@@H]2C[C@H]3[C@@H](O)C[C@@H](C2)N3C)=CC=CC=C1 WTQYWNWRJNXDEG-LEOABGAYSA-N 0.000 title claims abstract description 93
- 238000000034 method Methods 0.000 title claims abstract description 38
- -1 coumarin compound Chemical class 0.000 title description 8
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 title description 2
- 229960000956 coumarin Drugs 0.000 title description 2
- 235000001671 coumarin Nutrition 0.000 title description 2
- RODXRVNMMDRFIK-UHFFFAOYSA-N scopoletin Chemical compound C1=CC(=O)OC2=C1C=C(OC)C(O)=C2 RODXRVNMMDRFIK-UHFFFAOYSA-N 0.000 claims abstract description 86
- 238000002360 preparation method Methods 0.000 claims abstract description 79
- 239000000284 extract Substances 0.000 claims abstract description 50
- XEHFSYYAGCUKEN-UHFFFAOYSA-N Dihydroscopoletin Natural products C1CC(=O)OC2=C1C=C(OC)C(O)=C2 XEHFSYYAGCUKEN-UHFFFAOYSA-N 0.000 claims abstract description 44
- FWYIBGHGBOVPNL-UHFFFAOYSA-N scopoletin Natural products COC=1C=C2C=CC(OC2=C(C1)O)=O FWYIBGHGBOVPNL-UHFFFAOYSA-N 0.000 claims abstract description 44
- 229930000680 A04AD01 - Scopolamine Natural products 0.000 claims abstract description 43
- STECJAGHUSJQJN-GAUPFVANSA-N Hyoscine Natural products C1([C@H](CO)C(=O)OC2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 STECJAGHUSJQJN-GAUPFVANSA-N 0.000 claims abstract description 43
- STECJAGHUSJQJN-UHFFFAOYSA-N N-Methyl-scopolamin Natural products C1C(C2C3O2)N(C)C3CC1OC(=O)C(CO)C1=CC=CC=C1 STECJAGHUSJQJN-UHFFFAOYSA-N 0.000 claims abstract description 43
- 229960002646 scopolamine Drugs 0.000 claims abstract description 43
- STECJAGHUSJQJN-FWXGHANASA-N scopolamine Chemical compound C1([C@@H](CO)C(=O)O[C@H]2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 STECJAGHUSJQJN-FWXGHANASA-N 0.000 claims abstract description 40
- 238000000926 separation method Methods 0.000 claims abstract description 23
- 150000004775 coumarins Chemical class 0.000 claims abstract description 15
- 239000000287 crude extract Substances 0.000 claims abstract description 8
- 238000007670 refining Methods 0.000 claims abstract description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 54
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 34
- 239000002904 solvent Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 239000003480 eluent Substances 0.000 claims description 12
- 238000000825 ultraviolet detection Methods 0.000 claims description 11
- 238000000605 extraction Methods 0.000 claims description 10
- 239000012535 impurity Substances 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 8
- 238000000874 microwave-assisted extraction Methods 0.000 claims description 8
- 238000012856 packing Methods 0.000 claims description 6
- 239000011347 resin Substances 0.000 claims description 5
- 229920005989 resin Polymers 0.000 claims description 5
- 239000011148 porous material Substances 0.000 claims description 4
- 241000208341 Hedera Species 0.000 claims description 3
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- 239000012528 membrane Substances 0.000 claims description 2
- 229930182470 glycoside Natural products 0.000 abstract description 28
- 150000001875 compounds Chemical class 0.000 abstract description 25
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- 235000019510 Long pepper Nutrition 0.000 description 15
- 240000003455 Piper longum Species 0.000 description 15
- 235000008534 Capsicum annuum var annuum Nutrition 0.000 description 7
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- 229910021641 deionized water Inorganic materials 0.000 description 7
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
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- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 241000722363 Piper Species 0.000 description 2
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- 239000000463 material Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 241000999524 Anisodus tanguticus Species 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
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- 235000016761 Piper aduncum Nutrition 0.000 description 1
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- 235000008184 Piper nigrum Nutrition 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 206010042674 Swelling Diseases 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
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- 238000004128 high performance liquid chromatography Methods 0.000 description 1
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- 230000002093 peripheral effect Effects 0.000 description 1
- 238000004237 preparative chromatography Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
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- 238000012353 t test Methods 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- XLRPYZSEQKXZAA-OCAPTIKFSA-N tropane Chemical compound C1CC[C@H]2CC[C@@H]1N2C XLRPYZSEQKXZAA-OCAPTIKFSA-N 0.000 description 1
- 229930004006 tropane Natural products 0.000 description 1
- 229930004668 tropane alkaloid Natural products 0.000 description 1
- 150000003813 tropane derivatives Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a method for separating and preparing coumarin compounds from anisodamine, belonging to the field of compound preparation. The method of the invention comprises the following steps: (1) preparing anisodamine extract; (2) refining the crude extract; (3) separation and purification: dissolving the anisodamine extract prepared in the step (2), injecting into a parallel separation preparation chromatograph, separating by a first-stage preparation chromatographic column, and separating by a second-stage preparation chromatographic column to obtain scopolamine, scopoletin and piperlongin. The invention successfully separates and prepares scopolamine, scopoletin and farnesoid glycoside from anisodamine. The preparation method has the advantages of simple process and operation, low cost, high yield and high purity of the prepared product, meets the requirement of being used as a chemical reference substance, and can be used as the chemical reference substance. The invention overcomes the defects of the prior art, makes up the blank of the prior art and has wide application prospect.
Description
Technical Field
The invention belongs to the field of compound preparation, and in particular relates to a method for separating and preparing coumarin compounds from anisodamine.
Background
Anisodamine Anisodus tanguticus (maxim.) Pascher, common name: tang Chuanna Bao, litsea camphorata, anisodamine yellow, qinghai, gansu, tibet (eastern), yunnan (northwest); it is grown on hillside and grass slope at 2800-4200 m altitude. The root is used for medicine and has analgesic effect; the plant is also an important resource plant for extracting tropane alkaloids; the aerial parts are mixed into cattle feed, and have the effect of promoting fat. Anisodamine, scopolamine and other tropane components are enriched in anisodamine, anisodamine and scopolamine, and the compound has obvious peripheral anticholinergic action, can resist the contraction of intestinal and bladder smooth muscle and the blood pressure drop caused by acetylcholine, and can reduce the intestinal tension in vivo. In addition, coumarin compounds in anisodamine such as scopoletin, and long-screen longleaf pepper branch glycoside have good biological activity, such as scopoletin has good antiinflammatory and antitumor effects, the method fructus Piperis Longi glycoside can be used for treating arthralgia due to wind-cold-dampness, nephritis, urosis, traumatic injury, swelling and pain, and fatigue etc., and scopolamine has antiinflammatory and antitumor effects.
However, the current separation method of coumarin compounds in anisodamine is less studied. The patent with the application number of 201210066768.X discloses a preparation method of scopolamine glycoside monomers in anisodamine roots, which adopts macroporous resin and semi-preparation chromatography to separate and prepare scopolamine glycosides, but the preparation method in the patent has complicated and complex separation steps and higher cost. There is no report about the separation and preparation of scopoletin and fasciatin from anisodamine.
Provides a method for preparing scopolamine, scopoletin and long-grating branch glycoside from anisodamine with high efficiency and low cost, and has important significance for researching coumarin compounds in anisodamine and applying the coumarin compounds in medicine.
Disclosure of Invention
The invention aims to provide a method for separating and preparing coumarin compounds from anisodamine. In particular to compounds scopolamine, scopoletin and piper longum glycoside which are simultaneously separated and extracted from anisodamine.
The invention provides a method for separating and preparing coumarin compounds from anisodamine, which comprises the following steps:
(1) Preparing anisodamine extract extractum: extracting anisodamine by adopting a microwave extraction method by adopting 10-85% alcohol solvent, and concentrating the extracting solution under reduced pressure to obtain anisodamine extract;
(2) Refining the crude extract: dissolving the anisodamine extract prepared in the step (1), removing impurities by using membrane separation equipment, and drying to obtain refined anisodamine extract;
(3) And (3) separating and purifying: dissolving the anisodamine extract prepared in the step (2), injecting the solution into a parallel separation preparation chromatograph, separating by a first-stage preparation chromatographic column, separating by a second-stage preparation chromatographic column, and separating to obtain three coumarin compounds; the coumarin compounds are scopolamine, scopoletin and farnesoid respectively.
Further, in the step (1), the alcohol solvent is ethanol or methanol; and/or in the step (1), the using amount of the alcohol solvent is 5-30 times of the anisodamine; and/or, in the step (1), during the microwave extraction, the microwave power is 300-1500W; and/or in the step (1), the extraction temperature is 40-70 ℃ and the extraction time is 20-60 min during the microwave extraction.
Further, in the step (1), the amount of the alcohol solvent is 10-20 times of that of anisodamine.
Further, in the step (2), the solvent for dissolving anisodamine extract is water; and/or in the step (2), the dosage of the solvent for dissolving the anisodamine extract is 1-30 times of the anisodamine extract; and/or, in the step (2), the impurity is removed by using a roll film, and the pore size of the roll film is 0.1-0.5 μm;
preferably, the pore size of the roll film is 0.2 μm to 0.5 μm.
Further, in the step (3), the solvent for dissolving anisodamine extract is water.
Further, in the step (3), when the first-stage preparation chromatographic column is separated, the eluent is methanol or ethanol with the volume concentration of 10-50%; and/or the flow rate of the eluent is 10-60 mL/min; and/or the ultraviolet detection wavelength is 200-400 nm.
Further, in the step (3), when the first-stage preparation chromatographic column is separated, the first-stage preparation chromatographic column filler is polar macroporous resin;
preferably, in step (3), the polar macroporous resin is of the type AB-8, D150, NKA or HJ05.
Further, in the step (3), before the separation of the secondary preparation chromatographic column, the components separated by the primary preparation chromatographic column are enriched and concentrated.
Further, in the step (3), when the secondary preparation chromatographic column is separated, the eluent is methanol or acetonitrile with the volume concentration of 10-30%; and/or the flow rate of the eluent is 5-60 mL/min; and/or the ultraviolet detection wavelength is 200-400 nm.
Further, in the step (3), when the secondary preparation chromatographic column is separated, the chromatographic packing in the secondary preparation chromatographic column is C18 or C8;
preferably, in step (3), the chromatographic packing material is of the type Unitary C18, C18HCE, xamide or helara C18.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for separating and preparing coumarin compounds from anisodamine. The preparation method has simple process and operation and low cost, and the prepared scopolamine, scopoletin and long-grating branch glycoside have high yield and purity, meet the requirements of being used as chemical reference substances, and can be used as the chemical reference substances. The invention overcomes the defects of the prior art, makes up the blank of the prior art and has wide application prospect.
The invention adopts the preparation chromatographic device with parallel separation mode to separate and prepare three chemical reference substances, which not only has simple process and high practicability, but also realizes the rapid separation and preparation of samples; meanwhile, on the premise of ensuring high-efficiency separation, the yield of chemicals of the sample is improved, and chemical reference substances with gram-grade purity of more than 98% can be obtained.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a diagram of the separation preparation of the secondary preparation of the preparative chromatography apparatus in parallel separation mode of example 1.
FIG. 2 is a graph showing the purity of the piper longi glycoside prepared in example 1.
FIG. 3 is a graph showing the purity of scopolamine prepared in example 1.
FIG. 4 is a graph showing the purity of scopoletin lactone prepared in example 1.
Detailed Description
The materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
Example 1 preparation of scopolamine, scopoletin and Falong from anisodamine
The method for preparing scopolamine, scopoletin and farnesoid glycoside from anisodamine comprises the following steps:
(1) Preparing anisodamine extract extractum:
extracting 200g anisodamine with 10 times of 75% ethanol under microwave power of 1200w at 60deg.C for 20min to obtain extractive solution; concentrating the extractive solution under reduced pressure to obtain anisodamine extract.
(2) Refining the crude extract:
dissolving anisodamine extract prepared in step (1) with 15 times of deionized water, pouring into a roll film, removing impurities with 0.3 μm roll film to obtain clear solution passing through the roll film, and drying the clear solution to obtain refined anisodamine extract.
(3) And (3) separating and purifying:
dissolving anisodamine extract prepared in the step (2) with water, injecting into a preparation chromatographic device with parallel separation mode, separating by a first-stage preparation chromatographic column with a packing D150, eluting with 30ml/min 30% methanol with a volume concentration, and ultraviolet detecting with a wavelength of 280nm to obtain the target compound component.
The target compound component is enriched and concentrated by a trapping device, then enters a secondary preparation chromatographic column, is separated by the secondary preparation chromatographic column, the secondary preparation chromatographic column is Unitary C18, and is eluted by methanol with the flow rate of 30ml/min and the volume concentration of 20 percent, and the ultraviolet detection wavelength is 280nm. According to the detected ultraviolet spectrum, respectively collecting chromatographic peaks, evaporating the solvent to dryness to respectively obtain 3 compounds (long pepper branch glycoside, scopoletin and scopoletin) which meet the requirements of chemical reference substances and have purity of more than 98%. The structure of the method piper longum glycoside isThe yield was 2.5%; the scopolamine has the structure +.>The yield was 3.7%; scopoletin has the structure +.>The yield thereof was found to be 2.3%.
Example 2 preparation of scopolamine, scopoletin and Falong from anisodamine
The method for preparing scopolamine, scopoletin and farnesoid glycoside from anisodamine comprises the following steps:
(1) Preparing anisodamine extract extractum:
extracting 1000g anisodamine with 15 times of 10% ethanol under microwave power of 1500w at 40deg.C for 30min to obtain extractive solution; concentrating the extractive solution under reduced pressure to obtain anisodamine extract.
(2) Refining the crude extract:
dissolving anisodamine extract prepared in step (1) with 15 times of deionized water, pouring into a roll film, removing impurities with 0.5 μm roll film to obtain clear solution passing through the roll film, and drying the clear solution to obtain refined anisodamine extract.
(3) And (3) separating and purifying:
dissolving anisodamine extract prepared in the step (2) with water, injecting into a preparation chromatographic device with parallel separation mode, separating by a first-stage preparation chromatographic column with the model of HJ05, eluting with methanol with the flow rate of 10ml/min and the volume concentration of 50%, and detecting the ultraviolet wavelength of 200nm to obtain the target compound component.
The target compound component is enriched and concentrated by a trapping device, then enters a secondary preparation chromatographic column, is separated by the secondary preparation chromatographic column, the model of the secondary preparation chromatographic column is Hedera C18, and is eluted by methanol with the flow rate of 60ml/min and the volume concentration of 30 percent, and the ultraviolet detection wavelength is 400nm. According to the detected ultraviolet spectrum, respectively collecting chromatographic peaks, evaporating the solvent to dryness to respectively obtain 3 compounds (long pepper branch glycoside, scopoletin and scopoletin) which meet the requirements of chemical reference substances and have purity of more than 98%. The yield of the piper longum glycoside is 2.2%; the yield of scopolamine is 3.0%; the yield of scopoletin was 2.9%.
Example 3 preparation of scopolamine, scopoletin and Falong from anisodamine
The method for preparing scopolamine, scopoletin and farnesoid glycoside from anisodamine comprises the following steps:
(1) Preparing anisodamine extract extractum:
extracting 3000g anisodamine with 20 times of 85% ethanol under microwave conditions of microwave power 300w and extraction temperature 40 deg.C for 40min to obtain extractive solution; concentrating the extractive solution under reduced pressure to obtain anisodamine extract.
(2) Refining the crude extract:
dissolving anisodamine extract prepared in step (1) with 15 times of deionized water, pouring into a roll film, removing impurities with 0.5 μm roll film to obtain clear solution passing through the roll film, and drying the clear solution to obtain refined anisodamine extract.
(3) And (3) separating and purifying:
dissolving anisodamine extract prepared in the step (2) with water, injecting into a preparation chromatographic device with parallel separation mode, separating by a first-stage preparation chromatographic column with the model of AB-8, eluting with methanol with the flow rate of 60ml/min and the volume concentration of 10%, and ultraviolet detecting with the wavelength of 400nm to obtain the target compound component.
The target compound component is enriched and concentrated by a trapping device, then enters a secondary preparation chromatographic column, is separated by the secondary preparation chromatographic column, the model of the secondary preparation chromatographic column is C18HCE, is eluted by methanol with the flow rate of 5ml/min and the volume concentration of 10 percent, the ultraviolet detection wavelength is 200nm, chromatographic peaks are respectively collected according to the detected ultraviolet spectrum, and 3 compounds (the long pepper branch glycoside, the scopoletin and the scopoletin lactone) which meet the requirements of chemical reference substances and have the purity of more than 98 percent are respectively obtained after the solvent is evaporated. The yield of the piper longum glycoside is 2.8%; the yield of scopolamine is 3.9%; the yield of scopoletin was 2.5%.
Example 4 preparation of scopolamine, scopoletin and Falong from anisodamine
The method for preparing scopolamine, scopoletin and farnesoid glycoside from anisodamine comprises the following steps:
(1) Preparing anisodamine extract extractum:
performing microwave extraction on 6000g anisodamine with 15 times of 65% ethanol (the microwave extraction condition is that the microwave power is 1000w, the extraction temperature is 50deg.C, and the extraction time is 60 min) to obtain extractive solution; concentrating the extractive solution under reduced pressure to obtain anisodamine extract.
(2) Refining the crude extract:
dissolving anisodamine extract prepared in step (1) with 10 times of deionized water, pouring into a roll film, removing impurities with 0.3 μm roll film to obtain clear solution passing through the roll film, and drying the clear solution to obtain refined anisodamine extract.
(3) And (3) separating and purifying:
dissolving anisodamine extract prepared in the step (2) with water, injecting into a preparation chromatographic device with parallel separation mode, separating by a first-stage preparation chromatographic column with NKA type, eluting with ethanol with flow rate of 30ml/min and volume concentration of 25%, and ultraviolet detection wavelength of 320nm to obtain target compound component.
The target compound component is enriched and concentrated by a trapping device, then enters a secondary preparation chromatographic column, is separated by the secondary preparation chromatographic column, the model of the secondary preparation chromatographic column is Hedera C18, and is eluted by acetonitrile with the flow rate of 25ml/min and the volume concentration of 20 percent, and the ultraviolet detection wavelength is 200nm. According to the detected ultraviolet spectrum, respectively collecting chromatographic peaks, evaporating the solvent to dryness to respectively obtain 3 compounds (long pepper branch glycoside, scopoletin and scopoletin) which meet the requirements of chemical reference substances and have purity of more than 98%. The yield of the piper longum glycoside is 2.2%; the yield of scopolamine is 4.0%; the yield of scopoletin was 2.1%.
Example 5 preparation of scopolamine, scopoletin and Falong from anisodamine
The method for preparing scopolamine, scopoletin and farnesoid glycoside from anisodamine comprises the following steps:
(1) Preparing anisodamine extract extractum:
extracting 1000g anisodamine with 15 times of 50% ethanol under microwave conditions of 800w microwave power and 45 deg.C for 45min to obtain extractive solution; concentrating the extractive solution under reduced pressure to obtain anisodamine extract.
(2) Refining the crude extract:
dissolving anisodamine extract prepared in step (1) with 10 times of deionized water, pouring into a roll film, removing impurities with 0.2 μm roll film to obtain clear solution passing through the roll film, and drying the clear solution to obtain refined anisodamine extract.
(3) And (3) separating and purifying:
dissolving anisodamine extract prepared in the step (2) with water, injecting into a preparation chromatographic device with parallel separation mode, separating by a first-stage preparation chromatographic column with the model D150, eluting with 20ml/min ethanol with the volume concentration of 20%, and ultraviolet detecting with the wavelength of 300nm to obtain the target compound component.
The target compound component is enriched and concentrated by a trapping device, then enters a secondary preparation chromatographic column, is separated by the secondary preparation chromatographic column, the model of the secondary preparation chromatographic column is Unitary C18, and is eluted by acetonitrile with the flow rate of 20ml/min and the volume concentration of 15 percent, and the ultraviolet detection wavelength is 300nm. According to the detected ultraviolet spectrum, respectively collecting chromatographic peaks, evaporating the solvent to dryness to respectively obtain 3 compounds (long pepper branch glycoside, scopoletin and scopoletin) which meet the requirements of chemical reference substances and have purity of more than 98%. The yield of the piper longum glycoside is 2.8%; the yield of scopolamine is 3.4%; the yield of scopoletin was 2.9%.
Comparative example 1
According to the method of Chinese patent CN102617674, scopoletin monomer is extracted from anisodamine, and the specific method is as follows:
(1) Pulverizing anisodamine root, sieving with 40 mesh sieve to obtain anisodamine root powder;
(2) 300g anisodamine root powder is extracted by hot reflux with 6000mL (20 times) ethanol solution with 75% volume concentration, and the reflux extraction conditions are as follows: extracting at 50deg.C for 1 time for 0.5 hr, filtering to obtain filtrate, and concentrating under reduced pressure to obtain extract;
(3) Dissolving the extract with 5 times of deionized water, and extracting with n-butanol to obtain extract;
(4) Eluting with deionized water on nonpolar macroporous resin (AB-8), and eluting with 40% ethanol solution for 10 times of column volume to obtain eluent containing scopolamine;
(5) Concentrating the eluent under reduced pressure to obtain crude scopolamine extract, dissolving the crude scopolamine extract with 3 times of methanol, loading on a silica gel column (200 meshes), and eluting with 3 times of column volume of chloroform/methanol mixed solution (the volume ratio of chloroform to methanol is 4:1) to obtain purified scopolamine eluent;
(6) Separating and purifying the purified scopolamine eluent by a semi-preparative liquid chromatograph, wherein the model of a chromatographic column is Unitary C18, the mobile phase is a mixed solution of methanol and water, the volume ratio of the methanol to the water is 15:85, the flow rate is 15min/min, and the ultraviolet detection wavelength is 280nm. Drying the eluent under reduced pressure to constant weight to obtain white powdery scopolamine monomer. The yield of scopolamine monomer is 0.8% and the purity is 96%.
Comparing the preparation method of the embodiment of the invention with the preparation method of the comparative example 1, the following is known: the scopolamine yield obtained in comparative example 1 is only 0.8%, the purity is only 96%, and the scopolamine yield is lower than that obtained by the preparation method of the invention; meanwhile, the preparation method of the comparative example 1 has more extraction steps and silica gel column chromatography steps than the preparation method of the embodiment of the invention, and the extraction and silica gel column chromatography have low efficiency, low experimental repeatability and no automation. Therefore, the preparation method provided by the embodiment of the invention is higher in efficiency, simpler, more convenient, faster and higher in repeatability.
In addition, the experimental result analysis of the preparation method of the embodiment of the invention and the preparation method of the comparative example 1 shows that only scopolamine glycoside compounds can be separated in the preparation method of the comparative example 1, and 3 compounds can be obtained in one-time separation of the preparation method of the embodiment of the invention, so that the invention has higher efficiency and innovation.
The beneficial effects of the present invention are demonstrated by specific test examples below.
Test example 1 test as chemical control
The three compound reference substances prepared by the embodiment of the invention are subjected to purity detection, uniformity detection, stability detection and fixed value detection.
1. And (3) purity detection: agilent high performance liquid chromatograph; diamond C18 (250 mm. Times.4.6 mm, 5 μm) column; mobile phase: acetonitrile-0.2% formic acid aqueous solution (volume ratio 15:85), flow rate: 1.0 mL/min; column temperature: 25 ℃; run time: 30min; detection wavelength: 200-400 nm. The purity of scopolamine prepared in the embodiment 1 of the invention is 99.3%, the purity of scopoletin is 98.7%, and the purity of long pepper is 99.2%.
2. And (3) uniformity test: according to standard sample guidance requirements, a random sequence repeated measurement method is adopted, 3 parts of samples of 0.5mg are weighed respectively after the sub-packaging of three compound samples are randomly extracted, 1.0mL of methanol is used for dissolution, the samples are analyzed according to a purity analysis method, data analysis is carried out by a variance analysis method, uniformity is calculated, the uniformity of scopoletin purity prepared in the embodiment 1 of the invention is 99.87%, the uniformity of scopoletin purity is 99.39%, the uniformity of piperlongum glycoside purity is 99.52%, and the uncertainty of uniformity is less than 0.01%, which indicates that the compound prepared by the invention is good as a reference substance.
3. Stability experiment: (1) accelerated stability testing: three compound samples were randomly extracted and stored at-20℃and 2 to 8℃and 20℃and 40℃and 60℃respectively. The samples were tested for purity analysis over a period of 6 days, and the fixed value results were the average of 2 determinations. The results are shown in tables 1-3, and the results show that the average purity of the samples of the three control products prepared by the embodiment of the invention is greater than 99% after the three control products are placed at 5 different temperatures for 6 days, and the samples meet the requirements.
TABLE 1 stability test results of scopoletin prepared by the invention
TABLE 2 stability test results of scopolamine prepared by the invention
TABLE 3 stability test results of Piper longum glycosides prepared by the method of the present invention
(2) Long-term stability test: and (5) examining the change of the characteristic value of the standard sample with time. The sample is stored in a refrigerator at the temperature of 4 ℃ for 2 years, the prepared scopolamine, scopoletin and long pepper glucoside samples are sampled and inspected once every 6 months, each sample is continuously sampled for 5 times through HPLC analysis, the purity is obtained through a peak area normalization method, the average value is a fixed value result, and the data are subjected to statistical analysis through t-test. The average value is the constant value result. The results are shown in Table 4.
As can be seen from table 4: through accelerated stability test and long-term stability test of the sample, the three control products are proved to have better stability.
TABLE 4 results of the long-term stability test of the three compounds prepared according to the invention
In conclusion, the invention provides a method for separating and preparing coumarin compounds from anisodamine, and the method successfully separates and prepares scopolamine, scopoletin and fascian from anisodamine. The preparation method has simple process and operation and low cost, and the prepared scopolamine, scopoletin and long-grating branch glycoside have high yield and purity, meet the requirements of being used as chemical reference substances, and can be used as the chemical reference substances. The invention overcomes the defects of the prior art, makes up the blank of the prior art and has wide application prospect.
Claims (10)
1. A method for separating and preparing coumarin compounds from anisodamine is characterized in that: it comprises the following steps:
(1) Preparing anisodamine extract extractum: extracting anisodamine by adopting a microwave extraction method by adopting 10-85% alcohol solvent, and concentrating the extracting solution under reduced pressure to obtain anisodamine extract;
(2) Refining the crude extract: dissolving the anisodamine extract prepared in the step (1), removing impurities by using membrane separation equipment, and drying to obtain refined anisodamine extract;
(3) And (3) separating and purifying: dissolving the anisodamine extract prepared in the step (2), injecting the solution into a parallel separation preparation chromatograph, separating by a first-stage preparation chromatographic column, separating by a second-stage preparation chromatographic column, and separating to obtain three coumarin compounds; the coumarin compounds are scopolamine, scopoletin and farnesoid respectively;
in the step (2), the solvent for dissolving anisodamine extract is water;
in the step (2), impurities are removed by using a roll film, wherein the pore size of the roll film is 0.1-0.5 mu m;
in the step (3), the solvent for dissolving anisodamine extract is water;
in the step (3), when the first-stage preparation chromatographic column is separated, eluting solution is methanol or ethanol with the volume concentration of 10-50%; the flow rate of the eluent is 10-60 mL/min; the ultraviolet detection wavelength is 200-400 nm;
in the step (3), during the separation of the secondary preparation chromatographic column, the eluent is methanol or acetonitrile with the volume concentration of 10-30%; the flow rate of the eluent is 5-60 mL/min; the ultraviolet detection wavelength is 200-400 nm.
2. The method according to claim 1, characterized in that: in the step (1), the alcohol solvent is ethanol or methanol; and/or in the step (1), the using amount of the alcohol solvent is 5-30 times of the anisodamine; and/or in the step (1), during the microwave extraction, the microwave power is 300-1500W; and/or in the step (1), during the microwave extraction, the extraction temperature is 40-70 ℃ and the extraction time is 20-60 min.
3. The method according to claim 2, characterized in that: in the step (1), the use amount of the alcohol solvent is 10-20 times of that of anisodamine.
4. The method according to claim 1, characterized in that: in the step (2), the dosage of the solvent for dissolving the anisodamine extract is 1-30 times of the anisodamine extract.
5. The method according to claim 1, characterized in that: the pore size of the roll film is 0.2-0.5 mu m.
6. The method according to claim 1, characterized in that: in the step (3), when the first-stage preparation chromatographic column is separated, the first-stage preparation chromatographic column filler is polar macroporous resin.
7. The method according to claim 1, characterized in that: in the step (3), the first-stage preparation chromatographic column packing is AB-8 and D150.
8. The method according to claim 1, characterized in that: in the step (3), before the separation of the secondary preparation chromatographic column, the components separated by the primary preparation chromatographic column are enriched and concentrated.
9. The method according to claim 1, characterized in that: in the step (3), when the secondary preparation chromatographic column is separated, the chromatographic packing in the secondary preparation chromatographic column is C18 or C8.
10. The method according to claim 1, characterized in that: in the step (3), the model of the chromatographic packing is Unitary C18, C18HCE, xamide or Hedera C18.
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| CN102617674A (en) * | 2012-03-15 | 2012-08-01 | 中国科学院西北高原生物研究所 | Preparation method of scopolin monomer in anisodus tanguticus root |
| CN105481921A (en) * | 2015-12-29 | 2016-04-13 | 中国科学院西北高原生物研究所 | Method for separating new compound from whin based on parallel separation type preparative chromatography |
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| CN112755041A (en) * | 2021-02-09 | 2021-05-07 | 中国药科大学 | Application of coumarin derivative in preparation of medicine for preventing and/or treating gout |
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| CN102617674A (en) * | 2012-03-15 | 2012-08-01 | 中国科学院西北高原生物研究所 | Preparation method of scopolin monomer in anisodus tanguticus root |
| CN105481921A (en) * | 2015-12-29 | 2016-04-13 | 中国科学院西北高原生物研究所 | Method for separating new compound from whin based on parallel separation type preparative chromatography |
| CN110003197A (en) * | 2019-04-23 | 2019-07-12 | 南京久安源环保科技有限公司 | A kind of extraction process of tropane alkaloids |
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