Method for preparing bulleyaconitine A by utilizing supercritical chromatography

Abstract

The invention relates to the technical field of natural product separation and purification. The invention provides a method for preparing bulleyaconitine A by utilizing supercritical chromatography, which comprises the following steps: analyzing and preparing high-purity bulleyaconitine A by utilizing a supercritical chromatography system and taking supercritical fluid as a mobile phase A, an organic solvent as a mobile phase B, a reversed-phase filler or silica gel as a stationary phase filled chromatographic column and PDA (Personal Digital Assistant) as a detector. The method provided by the invention has the advantages of simple and feasible process, high separation speed, high separation efficiency, small amount of used solvent, easy recovery, use of supercritical fluid as a mobile phase, environmental protection, stable obtaining of high-purity bulleyaconitine A, large single sample loading amount, and realization of large-scale industrial application.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, and particularly relates to a method for preparing bulleyaconitine A by using supercritical chromatography. Background Art

[0002] Bulleyaconitine A [(1 α ,6 α ,14 α ,16 β )tetrahydro-8,13,14-triol-20-ethyl-1,6,16-trimethoxy-4-methoxymethyl-8-acetoxy-14-(4'-p-methoxybenzyl)-aconitane], English name: bulleyaconitine A, molecular formula: C 35 H 49 NO 10 , molecular weight: 643.77, CAS number: 107668-79-1, the chemical structure is shown in Formula I, and it is a white crystal or crystalline powder.

[0003] Formula I.

[0004] In 1980, Kunming Institute of Botany, Chinese Academy of Sciences isolated bulleyaconitine A from the herb Duola in Western Yunnan (Aconitum bulleyanum Diels), which is used for analgesia and anti-inflammatory in Yunnan folk. In 1983, Shanghai Institute of Materia Medica, Chinese Academy of Sciences confirmed that bulleyaconitine A has obvious analgesic and anti-inflammatory effects. Bulleyaconitine A is currently the non-addictive analgesic drug with the best clinical efficacy and the largest difference between the effective dose and the toxic dose acting on the peripheral nervous system. Aconitum bulleyanum Diels) and isolated bulleyaconitine A. In 1983, Shanghai Institute of Materia Medica, Chinese Academy of Sciences confirmed that bulleyaconitine A has obvious analgesic and anti-inflammatory effects. Bulleyaconitine A is currently the non-addictive analgesic drug with the best clinical efficacy and the largest difference between the effective dose and the toxic dose acting on the peripheral nervous system.

[0005] Due to the limitations of the structure of this type of aconitane diterpenoid alkaloid itself, semi-synthesis and synthesis are only limited to the scope of academic research. Currently, bulleyaconitine A can only be isolated from plants of the genus Aconitum, such as Aconitum bulleyanum A. bulleyanum ), Aconitum crassicaule A. crassicaule ), Aconitum georgei A. georgei ), Aconitum transsectum A. tranddectum ), and Aconitum vilmorinianum A. vilmorrianum ) etc. In recent years, bulleyaconitine A has shown definite curative effects in chronic diseases and geriatric diseases, and its clinical promotion has been rapid, showing great market growth potential and competitiveness. Therefore, the rapid large-scale preparation of bulleyaconitine A is particularly important.

[0006] Chinese patents CN115894370A, CN117327013A, CN1054976A, CN101555227A, CN101830849A, CN102775349A, CN102924376A, CN104326981A, CN106008344A, CN107245054A, and prior art such as "Yuan Mei. Research on the crude drug and chemical constituents of Aconitum bulleyanum Diels [D]. Yunnan University of Traditional Chinese Medicine, 2012." all disclose separation and purification methods using the combination of column chromatography and recrystallization and high-speed countercurrent chromatography. However, these methods are time-consuming, costly, and have complex pretreatment, making it difficult to achieve efficient large-scale preparation of mesaconitine. Summary of the Invention

[0007] The object of the present invention is to provide a method for preparing mesaconitine using supercritical chromatography. The present invention overcomes the deficiencies of the prior art and provides an analytical and preparative method for mesaconitine using supercritical chromatography technology.

[0008] To achieve the above object of the invention, the present invention provides the following technical solutions: The present invention provides a method for preparing mesaconitine using supercritical chromatography, comprising the following steps: using a supercritical chromatography system, with a supercritical fluid as mobile phase A, an organic solvent as mobile phase B, and a reversed-phase packing or silica gel as the stationary phase to fill the chromatographic column, and a PDA as the detector to analyze and prepare high-purity mesaconitine.

[0009] Preferably, the type of the supercritical fluid is CO 2 , NO 2 or NH 3 .

[0010] Preferably, the type of the organic solvent is one or more of methanol, ethanol, acetonitrile, and isopropanol.

[0011] Preferably, the type of the reversed-phase packing is a polystyrene / divinylbenzene type reversed-phase resin, and the type of the silica gel is a phenylsilane-bonded silica gel.

[0012] Preferably, the detection wavelength of the detector is 190 - 800 nm.

[0013] Preferably, the analytical method comprises the following steps: (1) Dissolve the sample in an organic solvent to obtain a sample solution; (2)Set the parameters of the supercritical chromatography system as follows: flow rate 0.5 - 5 mL / min, detection wavelength 190 - 800 nm, column temperature 25 - 50 °C, BPR pressure 5 - 50 MPa, BPR temperature 30 - 60 °C, modifier ratio 2 - 60%, chromatographic column C4 / C8 / C18 / RP18 / ODS / silica (3.0 - 4.6 × 100 - 250 mm, 3 - 5 μm); (3)Inject the sample solution into an analytical or analytical semi-preparative supercritical chromatograph and record the chromatogram; The types of samples described in step (1) are crude extracts containing mesaconitine, refined products containing mesaconitine, or mesaconitine; The types of organic solvents described in step (1) are methanol and / or ethanol, and the sample concentration in the sample solution is 0.5 - 2 mg / mL; The amount of the sample solution used in step (3) is 3 - 20 μL.

[0014] Preferably, the preparation method includes the following steps: A. Dissolve the sample with an organic solvent to obtain a sample solution; B. Set the parameters of the supercritical chromatography system as follows: total flow rate 15 - 100 mL / min, detection wavelength 190 - 800 nm, column temperature 25 - 50 °C, BPR pressure 50 - 200 MPa, BPR temperature 30 - 60 °C, modifier ratio 5 - 60%, chromatographic column C4 / C8 / C18 / RP18 / ODS / silica (4.5 - 9.5 × 100 - 250 mm, 10 - 50 μm); C. Inject the sample solution into a preparative supercritical chromatograph, record the chromatogram and collect the mesaconitine fraction; D. Repeat steps A - C for continuous preparation; E. Concentrate the mesaconitine fraction under reduced pressure and dry it under vacuum to obtain mesaconitine.

[0015] Preferably, the sample in step A is a crude extract containing mesaconitine, and the preparation method of the crude extract containing mesaconitine includes the following steps: ① Extract the raw drug with an alcohol - aqueous solution to obtain an extract; ② After extracting the extract, discard the oil phase, concentrate it to obtain a crude extract; The alcohol - aqueous solution in step ① is a methanol and / or ethanol solution, the volume fraction of the alcohol solution in the alcohol - aqueous solution is 30 - 100%, the number of extractions is 2 - 4 times, and the extraction time for each time is 80 - 160 min; In step ②, dichloromethane and / or chloroform and / or ethyl acetate are used for extraction, and the number of extractions is 2 - 4 times.

[0016] Preferably, the organic solvent in step A is methanol or / and ethanol, and the sample concentration in the sample solution is 0.5 - 5 mg / mL; The dosage of the sample solution in step C is 50 - 1000 μL.

[0017] The present invention provides a method for preparing aconitine by supercritical chromatography, which comprises the following steps: using a supercritical chromatography system, with supercritical fluid as mobile phase A, organic solvent as mobile phase B, and reverse-phase packing or silica gel as the stationary phase to fill the chromatographic column, and PDA as the detector, to analyze and prepare high-purity aconitine. The present invention overcomes the deficiencies of the prior art, utilizes supercritical chromatography technology, and provides a method for analyzing and preparing aconitine. The method provided by the present invention has the advantages of simple and feasible process, fast separation speed, high separation efficiency, less solvent used and easy recovery, uses supercritical fluid as the mobile phase, is green and environmentally friendly, can stably obtain high-purity aconitine, has a large single sample loading amount, and can realize large-scale industrial application.

[0018] The present invention controls the analysis and preparation parameters through supercritical chromatography technology, and further improves the purity and analysis and preparation efficiency of aconitine. As shown by the test results of the examples, the present invention can obtain aconitine with a purity of more than 99.80% only by performing supercritical chromatography preparation on the crude extract containing aconitine. Description of the Drawings

[0019] Figure 1 It is the SFC chromatographic analysis chart of the refined product containing aconitine prepared in Example 1; Figure 2 It is the SFC chromatographic analysis or / and semi-preparation chart of the refined product containing aconitine prepared in Example 1; Figure 3 It is the SFC chromatographic preparation chart and preparation stacking chart of the refined product containing aconitine prepared in Example 1; Figure 4 It is the SFC chromatographic preparation chart and preparation stacking chart of the refined product containing aconitine prepared in Example 1; Figure 5 It is the SFC chromatographic analysis chart and chromatographic analysis or / and semi-preparation chart of the crude extract containing aconitine prepared in Example 2; Figure 6 It is the SFC chromatographic analysis chart and chromatographic analysis or / and semi-preparation chart of the extract containing aconitine prepared in Example 3; Figure 7 It is the SFC chromatographic preparation chart and preparation stacking chart of the extract containing aconitine prepared in Example 3; Figure 8 It is the high-performance liquid chromatogram of aconitine prepared in Examples 1, 2, and 3. Detailed Description of the Invention

[0020] The present invention provides a method for preparing aconitine by supercritical chromatography, which comprises the following steps: using a supercritical chromatography system, with a supercritical fluid as mobile phase A, an organic solvent as mobile phase B, a reversed-phase packing or silica gel as the stationary phase to fill the chromatographic column, and a PDA as the detector to analyze and prepare high-purity aconitine.

[0021] In the present invention, the type of the supercritical fluid is preferably CO 2 , NO 2 or NH 3 .

[0022] In the present invention, the type of the organic solvent is preferably one or more of methanol, ethanol, acetonitrile and isopropanol.

[0023] In the present invention, the type of the reversed-phase packing is preferably a polystyrene / divinylbenzene type reversed-phase resin, and the type of the silica gel is preferably a phenylsilane-bonded silica gel.

[0024] In the present invention, the detection wavelength of the detector is preferably 190 - 800 nm.

[0025] In the present invention, the analysis method preferably comprises the following steps: (1) Dissolving the sample with an organic solvent to obtain a sample solution; (2) Setting the parameters of the supercritical chromatography system as: 0.5 - 5 mL / min, detection wavelength 190 - 800 nm, column temperature 25 - 50 °C, BPR pressure 5 - 50 MPa, BPR temperature 30 - 60 °C, modifier ratio 2 - 60%, chromatographic column C4 / C8 / C18 / RP18 / ODS / silica (3.0 - 4.6 × 100 - 250 mm, 3 - 5 μm); (3) Injecting the sample solution into an analytical or analytical and semi-preparative supercritical chromatograph and recording the chromatogram; The type of the sample in step (1) is preferably a crude extract containing aconitine, a refined product containing aconitine or aconitine; The type of the organic solvent in step (1) is preferably methanol or / and ethanol, and the sample concentration in the sample solution is preferably 0.5 - 2 mg / mL; The dosage of the sample solution in step (3) is preferably 3 - 20 μL.

[0026] In the present invention, the preparation method comprises the following steps: A. Dissolving the sample with an organic solvent to obtain a sample solution; B. Set the parameters of the supercritical chromatography system as follows: total flow rate 15 - 100 mL / min, detection wavelength 190 - 800 nm, column temperature 25 - 50 °C, BPR pressure 50 - 200 MPa, BPR temperature 30 - 60 °C, modifier ratio 5 - 60%, chromatographic column C4 / C8 / C18 / RP18 / ODS / silica (4.5 - 9.5 × 100 - 250 mm, 10 - 50 μm); C. Inject the sample solution into the preparative supercritical chromatograph, record the chromatogram and collect the aconitine fraction; D. Repeat steps A - C for continuous preparation; E. Concentrate the aconitine fraction under reduced pressure and dry it under vacuum to obtain aconitine.

[0027] In the present invention, the sample in step A is preferably a crude extract containing aconitine, and the preparation method of the crude extract containing aconitine preferably includes the following steps: ① Extract the raw drug with an alcohol - aqueous solution to obtain an extract; ② After extracting the extract, discard the oil - like substances, and concentrate to obtain a crude extract; In step ①, the alcohol - aqueous solution is preferably a methanol and / or ethanol solution, the volume fraction of the alcohol solution in the alcohol - aqueous solution is preferably 30 - 100%, the number of extractions is preferably 2 - 4 times, and the extraction time for each time is preferably 80 - 160 min; In step ②, during extraction, dichloromethane and / or chloroform and / or ethyl acetate are preferably used, and the number of extractions is preferably 2 - 4 times.

[0028] In the present invention, the type of the organic solvent in step A is preferably methanol or / and ethanol, and the sample concentration in the sample solution is preferably 0.5 - 5 mg / mL; The dosage of the sample solution in step C is preferably 50 - 1000 μL.

[0029] In the present invention, after the crude extract is segmented by silica gel and concentrated, a refined product containing aconitine can be obtained; the eluent used for silica gel segmentation preferably includes a mixed solution of two or more organic solvents such as petroleum ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol, and ethanol.

[0030] The technical solutions provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

[0031] Example 1 Refined Product This example provides a method for analyzing and / or preparing high - purity aconitine using supercritical chromatography, which includes: (1) Aconitum bulleyanum A. bulleyanumThe rhizomes were crushed (80 - 100 mesh), added with 10 times the amount of 60% methanol solution, and refluxed with alcohol under reduced pressure at 60 °C for 3 times, with each alcohol extraction time being 2 h. The obtained alcohol extracts were combined and concentrated to a non-alcoholic taste to obtain the extract; (2) Add an equal volume of dichloromethane to the extract and extract 3 times. Combine the organic phases and concentrate under reduced pressure to dryness to obtain the crude extract containing aconitine; (3) Subject the crude extract to silica gel column chromatography for fractionation (the eluent is dichloromethane:methanol = 200:1), collect the aconitine fractions, and concentrate under reduced pressure to obtain the refined product containing aconitine; (4) Use supercritical chromatography for analysis and / or preparation ① Analysis: Instruments and conditions: Supercritical liquid chromatograph: Shimadzu supercritical liquid chromatographic system SFC-LC (DAD); Chromatographic column: Shim-pack GIST-HP (C18-AQ, 3 μm, 4.6×250 mm); Mobile phase: A - supercritical CO 2 fluid; B - modifier (methanol); Time program: B Conc. 2% (0 - 1 min) → 20% (1 - 8 min) → 20% (8 - 9 min) → 2% (9 - 10 min); Flow rate: 2.0 mL / min; Column temperature: 40 °C; Back pressure regulator (BPR): 50 MPa, 50 °C; Injection volume: 5 μL; Experimental procedure: Take an appropriate amount of the refined product containing aconitine, filter it through a 0.25 μm filter membrane and inject it into the supercritical chromatography for analysis, and record the chromatogram. The results are shown in the appendix Figure 1 .

[0032] ② Analysis or / and semi-preparation: Instruments and conditions: Supercritical liquid chromatograph: Shimadzu supercritical liquid chromatographic system SFC-LC (DAD); Chromatographic column: Shim-pack GIST-HP (C18-AQ, 3 μm, 4.6×250 mm); Mobile phase: A - supercritical CO 2 fluid; B - modifier (methanol); Time program: B Conc. 5% methanol, 7 min / needle Flow rate: 2.5 mL / min; Column temperature: 40 °C; Back pressure regulator (BPR): 50 MPa, 50 °C; Injection volume: 5 μL; Experimental procedure: Take an appropriate amount of the refined product containing aconitine, filter it through a 0.25 μm filter membrane and inject it into supercritical chromatography for analysis. Record the chromatogram and collect the aconitine fraction, and then obtain high-purity aconitine by concentration under reduced pressure. The results are shown in the appendix Figure 2 .

[0033] ③ Preparation: Instrument and conditions: Supercritical liquid chromatograph: Buchi SFC-50; Chromatographic column: Shim-pack GIST-HP (C4, 5 μm, 9.4×150 mm); Mobile phase: A - supercritical CO 2 fluid; B - modifier (methanol - 1.0% diethylamine); Time program: B Conc. 5% methanol - 1.0% diethylamine, 5 min / needle, overlapping injection 4 min / needle; Flow rate: 15 mL / min; Column temperature: 40 °C; Back pressure regulator (BPR): 100 MPa, 40 °C; Injection volume: 100 μL; Experimental procedure: Take an appropriate amount of the refined product containing aconitine, filter it through a 0.25 μm filter membrane and inject it into supercritical chromatography for analysis. Record the chromatogram and collect the aconitine fraction, and then obtain high-purity aconitine by concentration under reduced pressure. The results are shown in the appendix Figure 3 .

[0034] ④ Preparation: Instrument and conditions: Supercritical liquid chromatograph: Buchi SFC-50; Chromatographic column: Silica 5 μm, 9.4×150 mm; Mobile phase: A - supercritical CO2 fluid; B - modifier (methanol - 1.0% diethylamine); Time program: B Conc. 30% methanol - 1.0% diethylamine, 6 min / needle, overlapping injection 3 min / needle; Flow rate: 20 mL / min; Column temperature: 40 °C; Back pressure regulator (BPR): 100 MPa, 40 °C; Injection volume: 200 μL; Experimental procedure: Take an appropriate amount of the refined product containing mesaconitine, filter it through a 0.25 μm filter membrane and inject it into supercritical chromatography for analysis. Record the chromatogram and collect the mesaconitine fraction, and concentrate it under reduced pressure to obtain high-purity mesaconitine. The results are shown in the appendix Figure 4 。

[0035] Example 2 Crude extract

[0036] Analyze and semi-prepare mesaconitine according to the method of Example 1, the difference from Example 1 is only that: directly use the crude extract containing mesaconitine obtained by S2 for analysis or / and semi-preparation. The results are shown in the appendix Figure 5 。

[0037] Example 3 Extract

[0038] Analyze and semi-prepare mesaconitine according to the method of Example 1, the difference from Example 1 is only that: (1) Directly use the extract containing mesaconitine obtained by S1 for analysis or / and semi-preparation and preparation; (2) Preparation by S4-3: Chromatographic column: Silica 5 μm, 9.4×150 mm; Time program: B Conc. 30% methanol - 1.0% diethylamine, 6 min / needle, overlapping injection 3 min / needle; Flow rate: 20 mL / min; The results are shown in the appendix Figure 6 and 7 。

[0039] The purity analysis of mesaconitine prepared in Examples 1 to 3 is shown in the appendix Figure 8 。[The HPLC analysis method is: Chromatographic column: Agilent ZORBAX SB-C18 5μm 4.6×150 mm; Time program: B - acetonitrile 0→100% (1 - 12 min)→100% (15min)→0% (18 min), A - 0.1% formic acid; Column temperature: 30℃].

[0040] As can be seen from the above examples, the present invention provides a method for preparing mesaconitine using supercritical chromatography, which includes the following steps: using a supercritical chromatography system, with supercritical fluid as mobile phase A, organic solvent as mobile phase B, and reverse-phase packing or silica gel as the stationary phase to fill the chromatographic column, and PDA as the detector to analyze and prepare high-purity mesaconitine. The method provided by the present invention has a simple and feasible process, a fast separation speed, a high separation efficiency, a small amount of solvent used and easy recovery, uses supercritical fluid as the mobile phase, is green and environmentally friendly, can stably obtain high-purity mesaconitine, has a large single sample loading amount, and can realize large-scale industrial application.

[0041] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing aconitine A using supercritical chromatography, characterized in that: The method comprises the following steps: using a supercritical chromatography system, using a supercritical fluid as a mobile phase A, an organic solvent as a mobile phase B, a reverse phase filler or silica gel as a stationary phase to fill a chromatographic column, and using PDA as a detector to analyze and prepare high-purity aconitone A; The analysis method comprises the following steps: (1) Dissolving the sample in an organic solvent to obtain a sample solution; (2) Set the supercritical chromatography system parameters as follows: 0.5-5 mL / min, detection wavelength 190-800 nm, column temperature 25-50 °C, BPR pressure 5-50 MPa, BPR temperature 30-60 °C, modifier ratio 2-60%, column C4 / C8 / C18 / RP18 / ODS / silica (3.0-4.6 × 100-250 mm, 3-5 μm); (3) Inject the sample solution into an analytical or analytical and semi-preparative supercritical fluid chromatograph and record the chromatogram; The sample in step (1) is a crude extract containing aconitine A, a refined product containing aconitine A, or aconitine A; The type of the organic solvent in step (1) is methanol and / or ethanol, and the sample concentration in the sample solution is 0.5-2 mg / mL; The amount of the sample solution used in step (3) is 3-20 μL.

2. The method according to claim 1, characterized in that The type of the supercritical fluid is CO2, NO2 or NH3.

3. The method according to claim 2, characterized in that The organic solvent is one or more of methanol, ethanol, acetonitrile and isopropanol.

4. The method according to claim 3, characterized in that: The type of the reverse phase filler is a polystyrene / divinylbenzene type reverse phase resin, and the type of the silica gel is a phenylsilane bonded silica gel.

5. The method according to claim 4, characterized in that The detection wavelength of the detector is 190-800 nm.

6. The method according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: A. dissolving the sample in an organic solvent to obtain a sample solution; B. Set the supercritical chromatography system parameters as follows: total flow rate 15~100mL / min, detection wavelength 190~800nm, column temperature 25~50℃, BPR pressure 50~200MPa, BPR temperature 30~60℃, modifier ratio 5~60%, column C4 / C8 / C18 / RP18 / ODS / silica (4.5~9.5×100~250mm, 10~50μm); C. Inject the sample solution into a preparative supercritical chromatograph, record the chromatogram and collect the aconitine A fraction; D. Repeat steps A to C for continuous preparation; E. Concentrate the aconitine A fraction under reduced pressure and dry in vacuum to obtain aconitine A.

7. The method according to claim 6, characterized in that The sample in step A is a crude extract containing aconitine A, and the preparation method of the crude extract containing aconitine A comprises the following steps: ① Extracting the raw drug with an alcohol-water solution to obtain an extract; ② After extracting the extract, discard the oily matter and concentrate to obtain the crude extract; The alcohol aqueous solution in step ① is a methanol and / or ethanol solution, the volume fraction of the alcohol solution in the alcohol aqueous solution is 30-100%, the number of extractions is 2-4 times, and the time of each extraction is 80-160 minutes; The extraction in step ② uses dichloromethane and / or chloroform and / or ethyl acetate, and the number of extractions is 2 to 4 times.

8. The method according to claim 6, characterized in that The type of the organic solvent in step A is methanol and / or ethanol, and the sample concentration in the sample solution is 0.5-5 mg / mL; The amount of the sample solution in step C is 50-1000 μL.

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