CN110845461B - Large-scale preparation method and application of high-purity scopoletin lactone - Google Patents
Large-scale preparation method and application of high-purity scopoletin lactone Download PDFInfo
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Abstract
The invention establishes a new method for obtaining high-purity natural scopoletin and crystals from waste tobacco branches in a large scale, takes the waste tobacco branches as raw materials, obtains scopoletin with the purity of 99.94 percent by solvent extraction and separation and purification of an industrial-grade high performance liquid chromatography system, obtains scopoletin crystals and identifies the spatial structure of the scopoletin crystals, has stable and controllable extraction and separation methods, realizes large-scale production, and is widely applied to the fields of medicines, chemical engineering and the like.
Description
Technical Field
The invention relates to the technical field of processing and extracting tobacco branch products, in particular to a method for extracting and crystallizing high-purity scopoletin and application thereof.
Background
The tobacco is also called as Mesona chinensis Benth, is a traditional Chinese medicinal material, and the inorganic components of the tobacco comprise water and mineral elements; the organic components mainly comprise saccharides, alkaloids, heterocycles, pigments, phenols, terpenes, organic acids, lipids, alcohols, aldehydes and ketones, and the like. Wherein, the organic components in the tobacco have multiple functions of antibiosis, antivirus, antitumor, antioxidation activity, in-vivo free radical removal and the like.
Scopoletin, also called scopoletin, has a molecular formula of C10H8O4, is soluble in hot ethanol, slightly soluble in water or cold ethanol, and easily soluble in chloroform, belongs to free coumarin compounds, and widely exists in plants, wherein the content of scopoletin, belladonna and Angelica dahurica of Umbelliferae plants is higher in roots of the plants.
Scopoletin lactone has wide pharmacological activity, can regulate blood pressure, can be used for treating bronchial diseases and asthma, can regulate hormone balance, and can help to relieve anxiety and depression. Scopoletin lactone is widely used in agriculture due to its insecticidal, acaricidal and bacteriostatic activities, and research reports that scopoletin lactone has the effects of analgesia, anti-inflammation, blood fat reduction, spasmolysis, oxidation resistance, tumor resistance, hyperuricemia prevention and the like. In addition, scopoletin lactone as a phytohormone has growth regulating effect and activity of inducing plant resistance.
Most of the medicinal scopoletin is extracted from Umbelliferae plant radix Saposhnikoviae, herba Caulophylli, and Convolvulaceae plant caulis Erycibes and herba Erodii Seu Geranii. In addition, cortex Mori, tobacco leaf, and Acanthopanax sessiliflorus fruit can also be used. The plant extracts are mainly obtained by traditional methods such as steam distillation, organic solvent extraction, column chromatography and the like.
In addition, 2, 4-dihydroxy anisole is used as a raw material to react with 3, 3-diethoxy ethyl propionate, 6, 7-dimethoxy coumarin is used as a raw material to be synthesized through two-step reaction, and p-benzoquinone is used as a raw material to be synthesized by microwave heating, but the methods can not realize large-scale production and obtain high-purity scopoletin.
In the earlier stage, tobacco leaves are used as raw materials, and scopoletin is obtained by extraction, macroporous resin, normal phase silica gel column chromatography separation and thin layer chromatography, but the steps are complex and the method cannot be repeated. At present, a new method for separating and extracting scopoletin by taking waste tobacco branches as raw materials (reducing cost) and exploring large-scale obtained high-purity scopoletin and scopoletin crystals is developed, and a material foundation is laid for further research and development.
Disclosure of Invention
In order to solve the problems, the invention provides a scopoletin lactone extraction and crystallization method and application thereof, which are used for improving the extraction amount of active components of tobacco branches.
In a first aspect, the present invention provides a method for extracting scopoletin, the method comprising the steps of:
(1) pretreatment: drying the tobacco branch raw material, and carrying out superfine grinding treatment on the dried tobacco branch raw material to obtain tobacco branch superfine powder;
(2) water extraction: adding an extraction solvent into the tobacco branch superfine powder, and performing extraction operation at the temperature of 50-100 ℃; centrifuging the tobacco branch superfine powder after the extraction operation, and collecting the precipitate after the centrifugation; the extraction solvent in the step (2) is purified water;
(3) methanol extraction: adding an extraction solvent into the precipitate obtained in the step (2), performing extraction at the extraction temperature of 0-4 ℃, centrifuging the extract, collecting the centrifuged supernatant, and performing rotary evaporation, concentration and drying on the collected supernatant to obtain a methanol-extracted supernatant concentrated extract; the extraction solvent in the step (3) is 85-100% of methanol;
(4) extracting with acetone: adding an extraction solvent into the precipitate obtained in the step (3), extracting and extracting at the extraction temperature of 0-4 ℃, centrifuging the impregnated Extract, collecting the centrifuged supernatant, and performing rotary evaporation, concentration, drying and low-temperature freeze drying on the collected supernatant to obtain an acetone-extracted supernatant concentrated Extract, namely Scopoletin Extract (SE); the extraction solvent in the step (4) is 85-100% of acetone;
(5) chromatographic separation and component activity analysis:
(a) dissolving the SE obtained in the step (4) by using a 60% methanol-water solution to obtain a sample with the concentration of 0.20g/mL, and separating by using a DAC150 reversed phase preparation liquid phase, wherein the elution mode is as follows: 5% methanol-water solution for 10min, 5% -20% methanol-water solution for 15min, 40% -55% methanol-water solution for 15min, 55% methanol-water solution for 10min, 65% methanol-water solution for 15min, ultraviolet detection wavelength of 260nm, sample loading of 200mL, and flow rate of 600 mL/min;
(b) dissolving TA-13-2 with absolute ethyl alcohol to obtain a sample with the concentration of 0.25g/mL, separating by using a DAC150 normal phase preparation liquid phase, wherein the elution mode is 20% ethanol-n-hexane solution for 20min, 100% ethanol-n-hexane solution for 10min, the ultraviolet detection wavelength is 260nm, the sample loading amount is 200mL, and the flow rate is 600 mL/min;
(6) identification of scopoletin lactone crystal space structure
(a) Method for culturing scopoletin lactone crystal
And (c) taking about 0.1g of TA-13-2 obtained in the step (5) and (b), placing the TA-13-2 into a 1.5mL glass bottle, adding 1mL of 20% ethanol n-hexane solution, fully dissolving, and culturing for 2 days under the conditions that the temperature is 20 ℃ and the humidity is 48 ℃ to obtain the colorless needle crystal.
(b) Identification of scopoletin crystal space structure by single crystal diffractometer
Preferably, the ultrafine grinding treatment in the step (1) is to perform coarse grinding treatment on the dried tobacco branch raw material by using a grinder with a 50-80-mesh screen, and perform grinding treatment (300-500 meshes) on the ground tobacco branch raw material by using an ultrafine grinder to obtain the tobacco branch ultrafine powder.
Preferably, the centrifugation treatment in the step (2) means that the concentrated extract solution after the temperature reduction treatment is placed in a centrifuge bottle, the centrifugation temperature is 8-12 ℃, the centrifugation time is 0.5-1.5h, and the rotation speed is 3000-.
Preferably, the mass of the extraction solvent added in step (3) is 1.5 to 2.5 times the mass of the precipitate.
Preferably, the dipping extraction operation in the step (4) is stirring once every 0.5h, and the extraction time is 12-24 h.
Preferably, the temperature of the concentration and drying treatment in the step (3) is 60-70 ℃.
Preferably, the chromatographic column packing used for preparing the liquid phase in step (5) (a) is C18 with the diameter of 10 μm and the size of 150mm × 250mm, and the elution mode is as follows: 5% methanol-water solution for 10min, 5% -20% methanol-water solution for 15min, 40% -55% methanol-water solution for 15min, 55% methanol-water solution for 10min, 65% methanol-water solution for 15min, detection wavelength of 260nm, sample loading amount of 200mL, and flow rate of 600 mL/min.
Preferably, the chromatographic column packing used for preparing the liquid phase in step (5) (b) is silica gel with the diameter of 10 μm, the size is 150mm multiplied by 250mm, the elution mode is 20% ethanol-n-hexane solution for 20min, 100% ethanol-n-hexane solution for 10min, the detection wavelength is 260nm, the sample loading amount is 200mL, and the flow rate is 600 mL/min.
The invention provides a method for extracting and crystallizing scopoletin and application thereof, which comprises the steps of pretreating tobacco branch raw materials, sequentially carrying out three operations of water extraction, methanol extraction and acetone extraction on the pretreated tobacco branch raw materials, using high-polarity solvent purified water, methanol and acetone as extraction solvents, fully extracting scopoletin in the tobacco branch raw materials by superfine grinding, improving the yield of scopoletin, obtaining scopoletin crystals and identifying the spatial structure of the scopoletin crystals.
The foregoing is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clear and clear, and to implement the technical solutions according to the content of the description, the following is a preferred embodiment of the present invention.
Drawings
FIG. 1 is a schematic diagram showing the process scheme for the extraction and separation of scopoletin obtained in example 1 of the present invention;
FIG. 2a shows a chromatogram for separating the scopoletin lactone extract obtained in example 1, (5), (a) of the present invention, where TA-13 is scopoletin lactone, and 1, 2, and 3 are chromatograms obtained by selecting three times arbitrarily (the chromatograms are substantially the same, which indicates that the method is substantially stable);
FIG. 2b shows an HPLC chromatogram for purity analysis of scopoletin TA-13 obtained in example 1, (5), (a) of the present invention, where the purity of scopoletin is 96.76% by area normalization;
FIG. 2c shows a chromatogram for TA-13 purification of scopoletin obtained in example 1, (5), (b) of the present invention, where TA-13-2 is scopoletin, and 1, 2, and 3 are chromatograms obtained by selecting randomly three times (the chromatograms are substantially the same, which indicates that the method is substantially stable);
FIG. 2d shows an HPLC chromatogram for analyzing purity of scopoletin TA-13-2 obtained in example 1, (5), (b) of the present invention, where the purity of scopoletin is 99.94% by area normalization;
FIG. 3 shows a TA-13-2 mass spectrum obtained in example 1 of the present invention;
FIG. 4 shows TA-13-2 obtained in example 1 of the present invention13C-NMR spectrum (400M);
FIG. 5 shows TA-13-2 obtained in example 1 of the present invention1H-NMR spectrum (400M);
FIG. 6 is a schematic representation of the molecular structure of scopoletin lactone;
FIG. 7 is a structural diagram of the scopoletin crystal space: grey spheres represent C atoms (numbered C1, C2, C3, C4, C5, C6, C7, C8, C9 and C10 respectively), red spheres represent O atoms (numbered O1, O2, O3 and O4 respectively), and blue spheres represent H atoms (numbered H1, H2, H3, H4, H5, H6, H7 and H8 respectively);
the foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to make the technical solutions of the present invention practical in accordance with the contents of the description, the preferred embodiments of the present invention are described below.
Detailed Description
The present invention will be described in further detail with reference to examples. It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention provides a method for extracting active components from tobacco branches, which refers to a flow chart shown in figure 1 and can comprise the following steps:
1. the extraction steps of scopoletin lactone are as follows:
(1) and (4) pretreatment.
Weighing 30Kg of tobacco branch raw material, placing the tobacco branch raw material in an oven for drying at the drying temperature of 60 ℃ for 4 hours until the water content of the tobacco branches is less than 5%, and taking out the tobacco branches; taking the dried tobacco branch raw material, coarsely grinding the tobacco branch raw material by using a grinder with a 50-mesh screen, and placing the tobacco branch raw material into an ultrafine grinder to carry out ultrafine grinding treatment to obtain the tobacco branch ultrafine powder.
(2) And (5) water extraction.
Weighing 30Kg of tobacco branch superfine powder, adding into a 200L extraction tank, adding 100Kg of purified water, and stirring and extracting at 80 ℃ for 0.5 h; transferring the mixture into a centrifugal bottle after stirring and extracting, and centrifuging for 0.5h at 8 ℃ at the rotating speed of 4000 rpm/min; and collecting the precipitate after the centrifugal treatment.
(3) And (4) extracting by using methanol.
Adding the precipitate obtained in the step (2) into a stainless steel barrel, wherein each barrel contains 10Kg of the precipitate; 15Kg of methanol is added into each barrel, the mass fraction of the methanol is 92 percent, and the impregnation extraction operation is carried out at the extraction temperature of 0 ℃; wherein the extraction time is 12h, and the materials in the barrel are stirred once every 0.5 h; placing the extract in a centrifuge bottle, centrifuging at 3 deg.C for 1.5 hr at 4000 rpm/min; collecting centrifuged supernatant, transferring the collected supernatant to 50L rotary evaporator for concentration at 60 deg.C, transferring to baking pan after concentration, and baking at 60 deg.C to obtain methanol extract (TMS) of tobacco branch.
(4) And (4) extracting with acetone.
Adding the precipitate obtained in the step (3) into stainless steel barrels, wherein each barrel is 10 kg; adding 15kg of acetone into each barrel, wherein the mass fraction of the acetone is 95%, extracting at the extraction temperature of 4 ℃, wherein the extraction time is 12h, and stirring the substances in the barrels once every 0.5 h; placing the extract in a centrifuge bottle, centrifuging at 3 deg.C for 1.5h at 4000 rpm/min; collecting supernatant after centrifugation, transferring the collected supernatant to 50L rotary evaporation for concentration treatment at 60 ℃, transferring the supernatant to a baking pan after concentration, and baking the supernatant into extract at 60 ℃ to obtain SE.
(5) And (4) performing chromatographic separation.
(a) Dissolving the SE obtained in the step (4) by using a 60% methanol-water solution to obtain a sample with the concentration of 0.20g/mL, and separating by using a preparation liquid phase, wherein the elution mode is as follows: 5% methanol-water solution for 10min, 5% -20% methanol-water solution for 15min, 40% -55% methanol-water solution for 15min, 55% methanol-water solution for 10min, 65% methanol-water solution for 15min, detection wavelength of 260nm, sample loading of 200mL, flow rate of 600mL/min, the results are shown in FIG. 2 a.
(b) Dissolving the active component 12 obtained in the step (5) (a) by using absolute ethyl alcohol to obtain a sample with the concentration of 0.25g/mL, and separating by using a preparation liquid phase, wherein the elution mode is 20% ethanol-n-hexane solution for 20min, 100% ethanol-n-hexane solution for 10min, the detection wavelength is 260nm, the sample loading amount is 200mL, and the flow rate is 600mL/min, and the result is shown in figure 2 c.
(6) Structural analysis: and (b) taking the TA-13-2 obtained in the step (5) and (b) for mass spectrometry. Chromatographic conditions are as follows: 5-100% methanol-water solution for 10 min; 100% methanol for 5 min; mass spectrum conditions: positive ion mode primary mass spectrometry; reference ion mass to charge ratio:121.050873, and 922.009798. The ion mass spectrum of the compound is shown in figure 3,13the C-NMR spectrum is shown in FIG. 4,1the H-NMR spectrum is shown in FIG. 5, and the molecular structure is shown in FIG. 6.
(7) Identification of scopoletin lactone crystal space structure
(a) Method for culturing scopoletin lactone crystal
And (c) taking about 0.1g of TA-13-2 obtained in the step (5) and (b), placing the TA-13-2 into a 1.5mL glass bottle, adding 1mL of 20% ethanol n-hexane solution, fully dissolving, and culturing for 2 days under the conditions that the temperature is 20 ℃ and the humidity is 48 ℃ to obtain the colorless needle crystal.
(b) Identification of scopoletin spatial structure
Selecting 0.05 x 0.2mm scopoletin needle crystal under a microscope, identifying the space structure of the crystal by adopting a RAXIS-RAPID II single crystal diffractometer of Japan, selecting a detection voltage of 40kV, a current of 30mA, a temperature of 293K and an exposure time of 10s, adopting RAPID AUTO (Ver 3.3.1) operating software, radiating CuKa (lambda is 1.54187), collecting diffraction data in an omega scanning mode, wherein the data scanning range is 2 theta: 10.742-136.254 degrees, a data collection range of-8 ≦ h ≦ 7, -8 ≦ K ≦ 9, -10 ≦ l ≦ 10, and 4723 diffraction points are collected in a range of 10.744 ≦ 2 θ ≦ 136.29 degrees at a temperature of 293(2) K, wherein 1467 independent diffraction points (Rint 0.0396, Rsigma 0.0408) and 1183 observable points are collected. The data obtained are corrected by Lp factors and empirical coefficients, the crystal structure is solved by a direct method (SHELXL-97 program) and a Fourier synthesis method, the non-hydrogen atom coordinates, the anisotropic temperature and the factors are modified by a full matrix least square method based on F2, the hydrogen atoms are added by theoretical calculation, and finally the deviation factor R1 is 0.0428, and the deviation factor wR2 is 0.1122. The melting phenomenon of the crystal does not occur in the single crystal testing process, the data statistics result is shown in tables 1-6, and the data analysis software adopts OLEX2(Ver 1.2.6). The spatial structure is shown in FIG. 7
TABLE 1 Crystal data and structural parameters of single crystals
TABLE 2 partial atomic coordinates of single crystal (. times.10)4) And equivalent equidirectional displacement parameter
Ueq is defined as orthogonal U IJ1/3 of tensor trajectory
Table 3 single crystal anisotropy displacement parameters, anisotropy displacement factor index takes the following form: -2 π2[h2a*2U11+2hka*b*U12+...]
TABLE 4 Single Crystal bond Length data
TABLE 5 Single Crystal bond Angle data
TABLE 6 Hydrogen atom coordinates of single crystals
And isotropic displacement parameter
While the foregoing is directed to the preferred embodiment of the present invention, it is intended that all changes and modifications including those which come within the spirit and scope of the invention be embraced by the appended claims.
Claims (9)
1. A preparation method of scopoletin lactone is characterized by comprising the following steps:
(1) pretreatment: drying the tobacco branch raw material, and carrying out superfine grinding treatment on the dried tobacco branch raw material to obtain tobacco branch superfine powder;
(2) water extraction: adding an extraction solvent into the tobacco branch ultrafine powder, and extracting under a certain temperature condition; centrifuging the tobacco branch superfine powder after the extraction operation, and collecting the precipitate after the centrifugation; the extraction solvent in the step (2) is purified water;
(3) methanol extraction: adding an extraction solvent into the precipitate obtained in the step (2), performing immersion extraction at the extraction temperature of 0-4 ℃, performing centrifugal treatment on the immersion extract, collecting the supernatant after the centrifugal treatment, and performing rotary evaporation, concentration and drying treatment on the collected supernatant to obtain a methanol extraction supernatant concentrated extract (TMS); the extraction solvent in the step (3) is 85-100% of methanol;
(4) extracting with acetone: adding an extraction solvent into the precipitate obtained in the step (3), performing immersion extraction at an extraction temperature of 0-4 ℃, centrifuging the immersion extract, collecting the centrifuged supernatant, and performing rotary evaporation, concentration, drying and low-temperature freeze drying on the collected supernatant to obtain an acetone extraction supernatant of tobacco branches, namely Scopoletin Extract (SE); the extraction solvent in the step (4) is 85-100% of acetone;
(5) and (3) chromatographic separation:
(a) dissolving the SE obtained in the step (4) by using a 60% methanol-water solution to obtain a sample with the concentration of 0.2g/mL, and separating by using a DAC150 reverse phase preparation liquid phase, wherein the elution mode is as follows: isocratic elution with 5% methanol-water solution, isocratic elution with 5% -20% methanol-water solution, isocratic elution with 40% -55% methanol-water solution, isocratic elution with 65% methanol-water solution, ultraviolet detection wavelength of 260nm to obtain scopoletin with purity of 96.76%, and numbering is TA-13;
the purity analysis method of TA-13 comprises the following steps: dissolving TA-13 with anhydrous ethanol, filtering with 0.22 μm nylon microporous membrane to obtain sample, analyzing with analytical HPLC (high performance liquid chromatography), Silica, 5 μm4.6 × 250mm for purity, using n-hexane (A) and anhydrous ethanol (B) as mobile phase, loading 20 μ L, flow rate of 1mL/min, ultraviolet detection wavelength of 260nm, elution method of 100% A5min, 100% A-100% B30min, and 100% B5 min;
(b) dissolving TA-13 with anhydrous ethanol to obtain a sample with a concentration of 0.25g/mL, separating with DAC150 normal phase preparation liquid phase, wherein the elution mode is 20% ethanol-n-hexane solution isocratic elution, 100% n-hexane isocratic elution, and the ultraviolet detection wavelength is 260nm to obtain scopoletin with a purity of 99.94%, and the number is TA-13-2;
the purity analysis method of TA-13-2 comprises the following steps: dissolving TA-13-2 with anhydrous ethanol, filtering with 0.22 μm nylon microporous membrane to obtain sample, analyzing with analytical HPLC (Silica, 5 μm4.6 x 250 mm) for purity, using n-hexane (A) and anhydrous ethanol (B) as mobile phase, loading 20 μ L, flow rate of 1mL/min, ultraviolet detection wavelength of 260nm, and elution method of 100% A5min, 100% A-100% B30min, and 100% B5 min.
2. The method of preparing scopoletin according to claim 1, wherein: the preparation method of the scopoletin lactone further comprises the following steps:
(a) method for culturing scopoletin lactone crystal
Placing the TA-13-20.1g obtained in the step (5) (b) in a 1.5mL glass bottle, adding 1mL of 20% ethanol-n-hexane solution for full dissolution, and culturing for 2 days at 20 ℃ and 48 ℃ humidity to obtain colorless needle crystals;
(b) identification of scopoletin lactone spatial structure
Selecting 0.05 x 0.2mm scopoletin needle crystal under microscope,
the spatial structure of the crystal is identified by adopting a RAXIS-RAPID II single crystal diffractometer of Japan science company, the detection voltage is selected to be 40kV, the current is 30mA, the temperature is 293K, the exposure time is 10s, RAPID AUTO operating software is adopted, CuK alpha, lambda is 1.54187 radiation, diffraction data are collected in an omega scanning mode, and the data scanning range is 2 theta: 10.742-136.254 degrees, the data collection range is-8 is more than or equal to h and less than or equal to 7, -8 is more than or equal to K and less than or equal to 9, -10 is more than or equal to l and less than or equal to 10, 4723 diffraction points are collected in the range of 10.744 degrees and more than or equal to 2 theta and less than or equal to 136.29 degrees at the temperature of 293K, wherein 1467 independent diffraction points, Rint 0.0396, Rsigma 0.0408 and 1183 observable points are collected; the obtained data is corrected by Lp factor and empirical coefficient, the crystal structure is solved by direct method, SHELXL-97 program and Fourier synthesis method, the non-hydrogen atom coordinate, anisotropic temperature and factor are modified by full matrix least square method based on F2, the hydrogen atom is added by theoretical calculation, and finally the deviation factor R1 is 0.0428, and wR2 is 0.1122.
3. The method of preparing scopoletin according to claim 1, wherein the at least one compound selected from the group consisting of: the ultrafine grinding treatment in the step (1) is to perform coarse grinding treatment on the dried tobacco branch raw material by using a grinder with a screen of 50-80 meshes, and perform grinding treatment on the tobacco branch raw material after the coarse grinding treatment by using an ultrafine grinder with the screen of 300 meshes to obtain the tobacco branch ultrafine powder.
4. The method of preparing scopoletin according to claim 1, wherein the at least one compound selected from the group consisting of: the centrifugation temperature in the step (2) is 8-12 ℃, the centrifugation time is 0.5-1.5h, and the rotation speed is 3000-6000 rpm/min.
5. The method of preparing scopoletin according to claim 1, wherein: the mass of the extraction solvent added in the step (3) is 1.5-2.5 times of the mass of the precipitate.
6. The method of preparing scopoletin according to claim 1, wherein: the dipping and extracting operation in the step (3) is to stir once every 0.5h, and the extracting time is 12-24 h.
7. The method of preparing scopoletin according to claim 1, wherein the at least one compound selected from the group consisting of: the temperature of the concentration and drying treatment in the step (3) is 60-70 ℃.
8. The method of preparing scopoletin according to claim 1, wherein the at least one compound selected from the group consisting of: the chromatographic column packing used for preparing the liquid phase in the step (5) (a) is C18 with the diameter of 10 mu m, the size is 150mm multiplied by 250mm, and the elution mode is as follows: 5% methanol-water solution for 10min, 5% -20% methanol-water solution for 15min, 40% -55% methanol-water solution for 15min, 55% methanol-water solution for 10min, 65% methanol-water solution for 15min, detection wavelength of 260nm, sample loading of 200mL, and flow rate of 600 mL/min.
9. The method of preparing scopoletin according to claim 1, wherein the at least one compound selected from the group consisting of: the chromatographic column packing used for preparing the liquid phase in the step (5) (b) is Silica with the diameter of 10 mu m, the size is 150mm multiplied by 250mm, and the elution mode is as follows: the elution mode is 20min of 20% ethanol-n-hexane solution and 10min of 100% ethanol-n-hexane solution, the detection wavelength is 260nm, the sample loading amount is 200mL, and the flow rate is 600 mL/min.
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