CN114634539A - 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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- CN114634539A CN114634539A CN202210454236.7A CN202210454236A CN114634539A CN 114634539 A CN114634539 A CN 114634539A CN 202210454236 A CN202210454236 A CN 202210454236A CN 114634539 A CN114634539 A CN 114634539A
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- anisodamine
- extract
- scopoletin
- chromatographic column
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- 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 90
- 238000000034 method Methods 0.000 title claims abstract description 35
- -1 coumarin compound Chemical class 0.000 title abstract description 15
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 title description 3
- 235000001671 coumarin Nutrition 0.000 title description 3
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- RODXRVNMMDRFIK-UHFFFAOYSA-N scopoletin Chemical compound C1=CC(=O)OC2=C1C=C(OC)C(O)=C2 RODXRVNMMDRFIK-UHFFFAOYSA-N 0.000 claims abstract description 131
- XEHFSYYAGCUKEN-UHFFFAOYSA-N Dihydroscopoletin Natural products C1CC(=O)OC2=C1C=C(OC)C(O)=C2 XEHFSYYAGCUKEN-UHFFFAOYSA-N 0.000 claims abstract description 71
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- 239000000284 extract Substances 0.000 claims abstract description 58
- 238000000926 separation method Methods 0.000 claims abstract description 33
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- STECJAGHUSJQJN-USLFZFAMSA-N LSM-4015 Chemical compound C1([C@@H](CO)C(=O)OC2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 STECJAGHUSJQJN-USLFZFAMSA-N 0.000 description 1
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Images
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
Abstract
The invention provides a method for separating and preparing coumarin compounds from anisodamine, belonging to the field of compound preparation. The method 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 the mixture into a parallel separation preparative chromatograph, separating by a first-stage preparative chromatographic column, separating by a second-stage preparative chromatographic column, and separating to obtain scopoletin, scopoletin and fructus piperis longi glucoside. The invention successfully separates and prepares the scopoletin, the scopoletin lactone and the common piperlongumoside from the anisodamine. The preparation method has simple and convenient process and operation and low cost, and the prepared product has high yield and high purity, meets the requirements of serving as a chemical reference substance and can serve as the chemical reference substance. The invention overcomes the defects of the prior art, makes up the vacancy of the prior art and has wide application prospect.
Description
Technical Field
The invention belongs to the field of compound preparation, and particularly relates to a method for separating and preparing coumarin compounds from anisodamine.
Background
Anisodus tanguticus anserina anguticus (Maxim.) Pascher, trivial name: tangchuan Bao, Cinnamomum camphora, Hemerocallis citrina, Qinghai, Gansu, Tibet (east), Yunnan (northwest); is grown on hillside and grassy slope yang with the altitude of 2800 and 4200 meters. The root is used for medicine, and has analgesic effect; the plant is also an important resource plant for extracting the tropane alkaloid; the aerial part of the feed is mixed into cattle feed, and has effect of fattening. The anisodamine is rich in tropane components such as atropine, anisodamine, scopolamine and the like, has obvious peripheral anticholinergic effect, can resist contraction of intestinal and bladder smooth muscle and blood pressure reduction caused by acetylcholine, and can reduce intestinal tension in vivo. In addition, coumarin compounds in anisodamine such as scopoletin, and fructus Piperis Longi glycoside also have good bioactivity, such as scopoletin has good antiinflammatory and antitumor effects, and can be used for treating rheumatism, nephritis, astringent, traumatic injury, and fatigue, and scopoletin has antiinflammatory and antitumor effects.
However, few studies have been made on methods for separating coumarins from anisodamine. Patent application No. 201210066768.X discloses a preparation method of scopoletin monomer in anisodamine root, which adopts macroporous resin and semi-preparative chromatography to separate and prepare scopoletin, but the preparation method in the patent has complicated and complicated separation steps and higher cost. No related technical report exists for separating and preparing scopoletin and fructus piperis longi glucoside from anisodamine.
Provides a method for preparing scopoletin, scopoletin and fructus piperis longi glucoside from anisodamine with high efficiency and low cost, and has important significance for the research and medical application of coumarin compounds in anisodamine.
Disclosure of Invention
The invention aims to provide a method for separating and preparing coumarin compounds from anisodamine. In particular to a method for simultaneously separating and extracting scopoletin, scopoletin and piperlongumoside from anisodamine.
The invention provides a method for separating and preparing coumarin compounds from anisodamine, which comprises the following steps:
(1) preparing an anisodamine extract: extracting the anisodamine by adopting a microwave extraction method by adopting a 10-85% alcohol solvent, and concentrating an extracting solution under reduced pressure to obtain an anisodamine extract;
(2) refining the crude extract: dissolving the anisodamine extract prepared in the step (1), removing impurities by adopting membrane separation equipment, and drying to obtain a refined anisodamine extract;
(3) separation and purification: dissolving the anisodamine extract prepared in the step (2), injecting the dissolved anisodamine extract into a parallel separation preparative chromatograph, separating the anisodamine extract by a first-stage preparative chromatographic column, and separating the anisodamine extract by a second-stage preparative chromatographic column to obtain three coumarin compounds; the coumarin compounds are scopoletin, scopoletin and fructus Piperis Longi glycoside respectively.
Further, in the step (1), the alcohol solvent is ethanol or methanol; and/or in the step (1), the amount of the alcohol solvent is 5-30 times of that of the anisodamine; and/or in the step (1), during 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 microwave extraction.
Further, in the step (1), the amount of the alcohol solvent is 10-20 times of that of the anisodamine.
Further, in the step (2), the solvent for dissolving the 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 removal is to remove impurities by using a roll-up membrane, and the pore size of the roll-up membrane is 0.1-0.5 μm;
preferably, the pore size of the roll-type membrane is 0.2-0.5 μm.
Further, in the step (3), the solvent for dissolving the anisodamine extract is water.
Further, in the step (3), during the first-stage preparative chromatographic column separation, 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 preparative chromatographic column is separated, the filler of the first-stage preparative chromatographic column is polar macroporous resin;
preferably, in step (3), the polar macroporous resin is AB-8, D150, NKA or HJ 05.
Further, in the step (3), before the separation by the second-stage preparative chromatographic column, the components separated by the first-stage preparative chromatographic column are concentrated.
Further, in the step (3), during the separation of the secondary preparative chromatographic column, 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 preparative chromatographic column is used for separation, the chromatographic packing in the secondary preparative chromatographic column is C18 or C8;
preferably, in step (3), the chromatography packing material is of the type of Unit C18, C18HCE, Xamide or Hedera 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, which successfully separates and prepares scopoletin, scopoletin and fructus piperis longi glucoside from anisodamine at the same time. The preparation method has simple and convenient process and operation and low cost, and the prepared scopoletin, scopoletin and piperlongumoside have high yield and high purity, meet the requirements of serving 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 vacancy of the prior art and has wide application prospect.
The preparation chromatographic device with the parallel separation mode is used for separating and preparing the three chemical reference substances, so that the process is simple, the practicability is high, and the rapid separation and preparation of the sample are realized; meanwhile, on the premise of ensuring high-efficiency separation, the yield of chemicals of a sample is improved, and a chemical reference substance with gram-grade purity of more than 98% can be obtained.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a separation preparation diagram of the two-stage preparation of a preparative chromatography apparatus in parallel separation mode of example 1.
Fig. 2 is a graph showing purity of piper longumulomb prepared in example 1.
FIG. 3 is a chart showing the purity assay of scopolamine prepared in example 1.
FIG. 4 is a chart showing the purity assay of scopoletin prepared in example 1.
Detailed Description
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.
Example 1 preparation of scopoletin, scopoletin and fructus Piperis Longi glycoside from Anisodus tanguticus
The method for preparing scopoletin, scopoletin and fructus piperis longi glucoside from anisodamine comprises the following steps:
(1) preparing an anisodamine extract:
performing microwave extraction of 200g anisodamine with 10 times of 75% ethanol (microwave extraction conditions are: microwave power 1200w, extraction temperature 60 deg.C, extraction time 20min) to obtain extractive solution; concentrating the extract under reduced pressure to obtain anisodamine extract.
(2) Refining the crude extract:
dissolving the anisodamine extract prepared in the step (1) with 15 times of deionized water, injecting into a roll type membrane, removing impurities by adopting a 0.3 mu m roll type membrane to obtain a clear solution penetrating through the roll type membrane, and drying the clear solution to obtain the refined anisodamine extract.
(3) Separation and purification:
dissolving the anisodamine extract prepared in the step (2) with water, injecting into a preparative chromatographic device with a parallel separation mode, separating by a first-stage preparative chromatographic column with a filler D150, eluting with 30% methanol at a flow rate of 30ml/min and a volume concentration of 280nm at an ultraviolet detection wavelength, and thus obtaining a target compound component.
The target compound component is enriched and concentrated by a trapping device, then enters a secondary preparative chromatographic column, is separated by the secondary preparative chromatographic column, the secondary preparative chromatographic column is Unitry 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 280 nm. According to the detected ultraviolet spectrum, chromatographic peaks are respectively collected, and 3 compounds (the fabry perone long branch glycoside, the scopolamine 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 to dryness. The structure of common pepper twig glucoside is
The yield is 2.5%; the scopoletin has the structure of
The yield is 3.7%; the scopoletin lactone has the structure of
The yield thereof was found to be 2.3%.
Example 2 preparation of scopoletin, scopoletin and fructus Piperis Longi glycoside from Anisodus tanguticus
The method for preparing scopoletin, scopoletin and fructus piperis longi glucoside from anisodamine comprises the following steps:
(1) preparing an anisodamine extract:
extracting 1000g of anisodamine with 15 times of 10% ethanol under microwave extraction conditions (microwave power 1500w, extraction temperature 40 deg.C, extraction time 30min) to obtain extractive solution; concentrating the extract under reduced pressure to obtain anisodamine extract.
(2) Refining the crude extract:
dissolving the anisodamine extract prepared in the step (1) with 15 times of deionized water, injecting into a roll type membrane, removing impurities by adopting a 0.5 mu m roll type membrane to obtain a clear solution penetrating through the roll type membrane, and drying the clear solution to obtain the refined anisodamine extract.
(3) Separation and purification:
dissolving the anisodamine extract prepared in the step (2) with water, injecting the mixture into a preparative chromatographic device with a parallel separation mode, separating by using a first-stage preparative chromatographic column, wherein the model of the first-stage preparative chromatographic column is HJ05, eluting with methanol with the flow rate of 10ml/min and the volume concentration of 50%, and obtaining a target compound component with the ultraviolet detection wavelength of 200 nm.
The target compound component is enriched and concentrated by a trapping device, then enters a secondary preparative chromatographic column, is separated by the secondary preparative chromatographic column, the model of the secondary preparative 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 400 nm. According to the detected ultraviolet spectrum, chromatographic peaks are respectively collected, and 3 compounds (the fabry perone long branch glycoside, the scopolamine 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 to dryness. The yield of the fructus piperis longi glucoside is 2.2%; the yield of scopolamine is 3.0%; the yield of scopoletin lactone was 2.9%.
Example 3 preparation of scopoletin, scopoletin and fructus Piperis Longi glycoside from Anisodus tanguticus
The method for preparing scopoletin, scopoletin and fructus piperis longi glucoside from anisodamine comprises the following steps:
(1) preparing an anisodamine extract:
extracting 3000g of anisodamine with 20 times of 85% ethanol under microwave extraction conditions (microwave power 300w, extraction temperature 40 deg.C, extraction time 40min) to obtain extractive solution; concentrating the extract under reduced pressure to obtain anisodamine extract.
(2) Refining the crude extract:
dissolving the anisodamine extract prepared in the step (1) with 15 times of deionized water, injecting into a roll type membrane, removing impurities by adopting a 0.5 mu m roll type membrane to obtain a clear solution penetrating through the roll type membrane, and drying the clear solution to obtain the refined anisodamine extract.
(3) Separation and purification:
dissolving the anisodamine extract prepared in the step (2) with water, injecting into a preparative chromatographic device with a parallel separation mode, separating by a primary preparative 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 detecting with ultraviolet light at the wavelength of 400nm to obtain the target compound component.
The target compound components are enriched and concentrated by a trapping device, then enter a secondary preparative chromatographic column, and are separated by the secondary preparative chromatographic column, the model of the secondary preparative chromatographic column is C18HCE, and is eluted by methanol with the flow rate of 5ml/min and the volume concentration of 10%, the ultraviolet detection wavelength is 200nm, chromatographic peaks are respectively collected according to the detected ultraviolet spectrum, and after the solvent is evaporated to dryness, 3 compounds (namely the farnesyl glucoside, the scopolamine and the scopoletin lactone) which meet the requirements of chemical reference substances and have the purity of more than 98% are respectively obtained. The yield of the common piper longumleaf glycosides is 2.8%; the yield of scopolamine is 3.9%; the yield of scopoletin lactone was 2.5%.
Example 4 preparation of scopoletin, scopoletin and fructus Piperis Longi glycoside from Anisodus tanguticus
The method for preparing scopoletin, scopoletin and fructus piperis longi glucoside from anisodamine comprises the following steps:
(1) preparing an anisodamine extract:
extracting 6000g anisodamine with 15 times of 65% ethanol under microwave extraction conditions of 1000w microwave power, 50 deg.C extraction temperature and 60min extraction time to obtain extractive solution; concentrating the extract under reduced pressure to obtain anisodamine extract.
(2) Refining the crude extract:
dissolving the anisodamine extract prepared in the step (1) with 10 times of deionized water, injecting into a roll type membrane, removing impurities by adopting a 0.3 mu m roll type membrane to obtain a clear solution penetrating through the roll type membrane, and drying the clear solution to obtain the refined anisodamine extract.
(3) Separation and purification:
dissolving the anisodamine extract prepared in the step (2) with water, injecting the mixture into a preparative chromatographic device with a parallel separation mode, separating by a primary preparative chromatographic column with the model of NKA, eluting with 25% ethanol at the flow rate of 30ml/min and the volume concentration, and obtaining the target compound component with the ultraviolet detection wavelength of 320 nm.
The target compound component is enriched and concentrated by a trapping device, then enters a secondary preparative chromatographic column, is separated by the secondary preparative chromatographic column, the model of the secondary preparative 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 200 nm. According to the detected ultraviolet spectrum, chromatographic peaks are respectively collected, and 3 compounds (the fabry perone long branch glycoside, the scopolamine 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 to dryness. The yield of the fructus piperis longi glucoside is 2.2%; the yield of scopolamine is 4.0%; the yield of scopoletin lactone was 2.1%.
Example 5 preparation of scopoletin, scopoletin and fructus Piperis Longi glycoside from Anisodus tanguticus
The method for preparing scopoletin, scopoletin and fructus piperis longi glucoside from anisodamine comprises the following steps:
(1) preparing an anisodamine extract:
performing microwave extraction of 1000g anisodamine with 15 times of 50% ethanol (under microwave extraction conditions of 800w microwave power, 45 deg.C extraction temperature and 45min extraction time) to obtain extractive solution; concentrating the extract under reduced pressure to obtain anisodamine extract.
(2) Refining the crude extract:
dissolving the anisodamine extract prepared in the step (1) with 10 times of deionized water, injecting into a roll type membrane, removing impurities by adopting a 0.2 mu m roll type membrane to obtain a clear solution penetrating through the roll type membrane, and drying the clear solution to obtain the refined anisodamine extract.
(3) Separation and purification:
dissolving the anisodamine extract prepared in the step (2) with water, injecting into a preparative chromatographic device with a parallel separation mode, separating by a primary preparative chromatographic column with the model of D150, eluting with 20% ethanol at the flow rate of 20ml/min and the volume concentration, and detecting with ultraviolet light at 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 preparative chromatographic column, is separated by the secondary preparative chromatographic column with the model of Unitry C18, and is eluted by acetonitrile with the flow rate of 20ml/min and the volume concentration of 15%, and the ultraviolet detection wavelength is 300 nm. According to the detected ultraviolet spectrum, collecting chromatographic peaks respectively, and evaporating the solvent to dryness to obtain 3 compounds (farubicin, scopoletin and scopoletin) with purity of more than 98% meeting the requirements of chemical reference substances. The yield of the common piper longumleaf glycosides is 2.8%; the yield of scopolamine is 3.4%; the yield of scopoletin lactone 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 radix anisodame, and sieving with 40 mesh sieve to obtain radix anisodame powder;
(2) 300g of anisodamine root powder is extracted by hot reflux with 6000mL (20 times) of ethanol solution with 75 percent of volume concentration, and the reflux extraction conditions are as follows: extracting at 50 deg.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 extractive solution;
(4) passing the extractive solution through nonpolar macroporous resin (AB-8), eluting with deionized water, and eluting with 40% ethanol solution 10 times column volume to obtain scopoletin-containing eluate;
(5) concentrating the eluate under reduced pressure to obtain scopoletin crude extract, dissolving the crude extract with 3 times of methanol, loading on silica gel column (200 mesh), and eluting with chloroform/methanol mixed solution (the volume ratio of chloroform to methanol is 4:1)3 times of column volume to obtain purified scopoletin eluate;
(6) separating and purifying the purified scopoletin eluate with semi-preparative liquid chromatograph, wherein the model of chromatographic column is Unitry C18, the mobile phase is mixed solution of methanol and water, the volume ratio of methanol to water is 15:85, the flow rate is 15min/min, and the ultraviolet detection wavelength is 280 nm. Drying the eluent under reduced pressure to constant weight to obtain white powdered scopolamine monomer. The yield of the scopoletin 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 steps are carried out: the yield of the scopolamine prepared in the comparative example 1 is only 0.8%, the purity is only 96%, and the yield is lower than that of the scopolamine prepared by the preparation method; meanwhile, compared with the preparation method of the embodiment of the invention, the preparation method of the comparative example 1 has more extraction steps and silica gel column chromatography steps, and the extraction and silica gel column chromatography efficiency is low, the experimental repeatability is low, and the automation is not generated. Therefore, the preparation method provided by the embodiment of the invention has the advantages of higher efficiency, simplicity, convenience, rapidness and high 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 compounds can be separated by the preparation method of the comparative example 1, and 3 compounds can be separated by the preparation method of the embodiment of the invention at one time, so that the invention has high efficiency and innovation.
The advantageous effects of the present invention are demonstrated by specific test examples below.
Test example 1 examination of chemical control
The three compound reference substances prepared in the embodiment of the invention are subjected to purity detection, uniformity detection, stability detection and fixed value detection.
1. And (3) purity detection: an Agilent high performance liquid chromatograph; diamonsil C18(250 mm. times.4.6 mm, 5 μm) column; mobile phase: acetonitrile-0.2% aqueous formic acid (15: 85 by volume), flow rate: 1.0 mL/min; column temperature: 25 ℃; operating time: 30 min; detection wavelength: 200 to 400 nm. The purity of scopoletin prepared in example 1 of the invention is 99.3%, the purity of scopoletin is 98.7%, and the purity of fabperone is 99.2%.
2. And (3) uniformity inspection: according to the standard sample guidance requirement, a random sequence repeated measurement method is adopted, split-packaged samples of three compound samples are randomly extracted, 3 parts of 0.5mg sample are respectively weighed, 1.0mL of methanol is respectively used for dissolving, the samples are analyzed according to a purity analysis method, data analysis is carried out by an variance analysis method, and uniformity is calculated, so that the purity uniformity of the scopolamine prepared in the embodiment 1 of the invention is 99.87%, the purity uniformity of the scopoletin is 99.39%, the purity uniformity of the fabulophyllin is 99.52%, and uncertainty of the uniformity is less than 0.01%, which indicates that the compound prepared in the invention has good uniformity as a reference substance.
3. Stability test: (1) and (3) accelerated stability test: samples of the three compounds were randomly extracted and stored at-20 deg.C, 2-8 deg.C, 20 deg.C, 40 deg.C, and 60 deg.C, respectively. In the study, samples were examined by purity analysis for 6 days, and the fixed value results are the average of 2 measurements. The results are shown in tables 1-3, and the results show that the average purity of the samples obtained by placing the three reference substances prepared in the embodiment of the invention at 5 different temperatures for 6 days is more than 99%, and the samples meet the requirements.
TABLE 1 stability test results of scopoletin prepared by the present invention
Table 2. stability test results of scopolamine prepared by the invention
Table 3. stability test results of the Fapiperlongumine prepared by the invention
(2) Long-term stability test: the change of the characteristic value of the standard sample with time was examined. The sample is stored in a refrigerator at 4 ℃, the prepared scopoletin, scopoletin and fabperone samples are sampled and tested once every 6 months by taking 2 years as a period, each sample is continuously injected for 5 times through HPLC analysis, the purity is calculated by a peak area normalization method, the average value is a fixed value result, and the statistical analysis is carried out on the data by using t test. The average value is the fixed value result. The results are shown in Table 4.
As can be seen from Table 4: the three reference substances are proved to have better stability through the accelerated stability test and the long-term stability test of the sample.
TABLE 4 Long-term stability test results for three compounds prepared according to the invention
In conclusion, the invention provides a method for separating and preparing coumarin compounds from anisodamine, and the invention successfully separates and prepares scopoletin, scopoletin lactone and fangchinoline from anisodamine at the same time. The preparation method has simple and convenient process and operation and low cost, and the prepared scopoletin, scopoletin and piperlongumoside have high yield and high purity, meet the requirements of serving 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 vacancy 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: extracting the anisodamine by adopting a microwave extraction method by adopting a 10-85% alcohol solvent, and concentrating an extracting solution under reduced pressure to obtain an anisodamine extract;
(2) refining the crude extract: dissolving the anisodamine extract prepared in the step (1), removing impurities by adopting membrane separation equipment, and drying to obtain a refined anisodamine extract;
(3) separation and purification: dissolving the anisodamine extract prepared in the step (2), injecting the dissolved anisodamine extract into a parallel separation preparative chromatograph, separating the anisodamine extract by a first-stage preparative chromatographic column, and separating the anisodamine extract by a second-stage preparative chromatographic column to obtain three coumarin compounds; the coumarin compounds are scopoletin, scopoletin and fructus Piperis Longi glycoside respectively.
2. The method of claim 1, wherein: in the step (1), the alcohol solvent is ethanol or methanol; and/or in the step (1), the amount of the alcohol solvent is 5-30 times of that of the anisodamine; and/or in the step (1), during 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 microwave extraction.
3. The method of claim 2, wherein: in the step (1), the amount of the alcohol solvent is 10-20 times of that of anisodamine.
4. The method of claim 1, wherein: in the step (2), the solvent for dissolving the 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 removal is to remove impurities by using a roll-up membrane, and the pore size of the roll-up membrane is 0.1-0.5 μm;
preferably, the pore size of the roll-type membrane is 0.2-0.5 μm.
5. The method of claim 1, wherein: in the step (3), the solvent for dissolving the anisodamine extract is water.
6. The method of claim 1, wherein: in the step (3), when the first-stage preparative chromatographic column is used for separation, 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.
7. The method of claim 6, wherein: in the step (3), when the first-stage preparative chromatographic column is used for separation, the filler of the first-stage preparative chromatographic column is polar macroporous resin;
preferably, in step (3), the polar macroporous resin is AB-8, D150, NKA or HJ 05.
8. The method of claim 1, wherein: in the step (3), before the second-stage preparative chromatographic column separation, the components separated by the first-stage preparative chromatographic column are concentrated.
9. The method of claim 1, wherein: in the step (3), when the secondary preparation chromatographic column is used for separation, 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.
10. The method of claim 9, wherein: in the step (3), when the secondary preparative chromatographic column is used for separation, the chromatographic packing in the secondary preparative chromatographic column is C18 or C8;
preferably, in step (3), the chromatography packing material is of the type of Unit 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 |
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