CN115385928A - Method for separating and preparing pyrone compound in Tibetan capillary - Google Patents

Method for separating and preparing pyrone compound in Tibetan capillary Download PDF

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Publication number
CN115385928A
CN115385928A CN202211030057.7A CN202211030057A CN115385928A CN 115385928 A CN115385928 A CN 115385928A CN 202211030057 A CN202211030057 A CN 202211030057A CN 115385928 A CN115385928 A CN 115385928A
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column
separating
methanol
tibetan
preparing
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黄发霞
余国民
王林萍
刘兴亮
徐德芳
李文林
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Qinghai University
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Qinghai University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems

Abstract

The invention discloses a separation and preparation process of a novel pyrone compound in Tibetan capillaris. The specific preparation process comprises the following steps: the method comprises four steps of extraction, medium-pressure chromatography rough separation of a microporous resin column, preparation of a reversed-phase preparative column and purification of the reversed-phase preparative column. The extraction solvent, the microporous resin column, the reverse phase chromatographic column separation solvent and the reverse phase chromatographic separation material in the preparation process can be recycled; the raw material source is wide, the large-scale operation can be realized through the technological steps of cold soaking extraction of methanol at room temperature and the like, and the purity of the product can be ensured to be more than 95% through high-pressure preparative chromatographic separation.

Description

Method for separating and preparing pyrone compound in Tibetan capillary
Technical Field
The invention relates to the technical field of natural product separation, in particular to a separation and preparation method of a novel pyrone compound in Tibetan capillaris.
Background
Tibetan capillary artemisia (Swertia mussotii), an annual herbaceous plant of Swertia (Swertia) of Gentianaceae (gentiana), is a traditional alpine Tibetan medicine, and has the Tibetan medicine name: "Tida" is mainly distributed in Tibet, yunnan, northwest of Sichuan and southwest of Qinghai in China. As one of the eight delicacies of Tibetan medicine, it is commonly used for treating acute and chronic hepatitis, chronic cholecystitis, etc.
The main chemical components of the plant are ketones, flavonoids, iridoids, triterpenes and the like, and the plant has various pharmacological activities of protecting liver and gallbladder, resisting oxidation, reducing blood sugar, resisting inflammation, protecting heart and cerebral vessels and the like. The Tibetan capillary artemisia has rich resources and has better medicinal value and new drug development prospect. The Tibetan capillary artemisia tablet, the Gantaishu capsule and the flu pill which are researched and developed at present are all used by Tibetan capillary artemisia, so that in order to further accelerate the quality evaluation, the production and the marketing of Tibetan capillary artemisia and the research and development steps of related new drugs, the potential active ingredients of the Tibetan capillary artemisia are necessarily mined from the Tibetan capillary artemisia, and the Tibetan capillary artemisia tablet, the Gantaishu capsule and the flu pill have important significance for fully developing and utilizing the medicinal material resources of the Tibetan capillary artemisia.
Disclosure of Invention
The invention aims to provide a separation and preparation process of a novel pyrone compound in Tibetan capillary, which has high product purity and can realize large-scale production.
In order to realize the purpose of the invention, the following technical scheme is adopted:
a method for separating and preparing pyrone compounds from Tibetan capillary comprises the following steps:
step 1, extraction: drying Tibetan herba Artemisiae Scopariae in the shade, coarse pulverizing, extracting with methanol, filtering, mixing filtrates to obtain filtrate A, mixing filtrate A with silica gel, and drying under reduced pressure to obtain Tibetan herba Artemisiae Scopariae extract sample;
step 2, carrying out medium-pressure chromatography rough separation on the microporous resin column: mixing the extract of Tibetan herba Artemisiae Scopariae with sample, separating by medium pressure chromatography with microporous resin, detecting by ultraviolet detector, collecting the first main chromatographic peak fraction in the chromatogram, and drying under reduced pressure to obtain crude isolate Fr1 containing target components;
step 3, preparing a reversed-phase preparation column: adding methanol with volume concentration of 70-90% into the crude isolate Fr1 to dissolve to prepare a sample solution, filtering by a microporous filter membrane of 0.45 mu m to obtain a filtrate B containing a target component, separating the obtained filtrate B by a C18 reversed phase preparative column, detecting by an ultraviolet detector, collecting a corresponding first chromatographic peak fraction in a preparative chromatogram, and drying under reduced pressure to obtain a crude isolate Fr1-1 containing the target component;
and 4, reversed-phase preparative column purification: adding methanol into the crude isolate Fr1-1 to dissolve to prepare a sample solution, filtering by a 0.45 mu m microporous filter membrane to obtain a filtrate C containing a target component, separating the filtrate C by a C18 reversed-phase preparation column, detecting by an ultraviolet detector, collecting a corresponding first chromatographic peak fraction in a preparative chromatogram, and drying under reduced pressure to obtain an isolate Fr1-1-1, namely a new pyrone compound, which is named as Swermussina.
In the step 1, methanol is extracted, and the ratio of material to liquid is 1g: extracting 5 to 100mL for 2~4 times at room temperature for 6 to 8h each time.
In the step 1, the mass ratio of the silica gel to the Tibetan capillaris extract is 3:1.
In the step 3, methanol is added into the crude isolate Fr1 to prepare a sample solution with the concentration of 50.0-100.0 mg/mL.
In the step 4, methanol is added into the crude isolate Fr1-1 to prepare a sample solution with the concentration of 50.0-100.0 mg/mL.
In the steps 1 to 4, the conditions of reduced pressure drying are as follows: the vacuum degree is 50 to 250mbar, and the temperature is 40 to 60 ℃; the detection wavelength of the ultraviolet detector is 210nm.
In the step 2, the specification of the medium-pressure chromatographic column is as follows: the length of the column is 460mm, the diameter is 15 to 49mm, and the stationary phase of the microporous resin is CHP20P; the mobile phase A is water, the mobile phase B is methanol, the mobile phase C is dichloromethane, the chromatographic conditions are 0 to 150min,0 to 100 percent of B,150 to 300min,0 to 100 percent of C, the sample volume is 5 to 50g, and the flow rate is 8 to 57mL/min.
In the step 3, the specification of the C18 reversed phase preparation column is as follows: the column length is 250mm, the diameter is 20mm, and the granularity is 5 mu m; the mobile phase A is 0.1 percent trifluoroacetic acid-water solution, the mobile phase B is methanol solution, elution is carried out by 8%B in 0 to 30min, the sample injection volume is 0.2 to 1.0mL, and the flow rate is 19mL/min.
In the step 4, the specifications of the C18 reversed phase preparation column are as follows: the column length was 250mm, the diameter was 20mm, the particle size was 5 μm, the mobile phase A was a 0.1% trifluoroacetic acid-water solution, the mobile phase B was a methanol solution, and the elution was carried out according to the following equation (0 to 30min, 11%) B, the sample introduction volume was 0.2 to 1.0mL, and the flow rate was 19mL/min.
The invention has the beneficial effects that:
(1) The invention has low cost and high product purity
The solvent used for extraction, the solvent used for separation by the microporous resin column and the reversed phase chromatographic column can be recycled; materials used in the reversed-phase chromatographic separation can be recycled, the recycled solvent and the recycled separation materials ensure lower average separation cost, and the high-pressure preparative chromatographic separation can ensure that the purity of the product is more than 95 percent.
(2) The preparation method can meet the requirement of large-scale production
The raw material requirement is not high, the cost is low, the wild or commercially available Tibetan capillaris can be used, and the batch preparation is easy; the methanol is extracted by cold leaching at room temperature, and the operation is easy; the separation adopts coarse separation by a microporous resin column, and the microporous resin separation material can be arranged in a medium-pressure column chromatography system, so that large-scale production is easy to realize; the reversed phase preparative liquid chromatography used in the separation and purification is a rapid isocratic method, and is also very suitable for mass production.
Drawings
FIG. 1 is a medium pressure chromatographic separation diagram of a sample mixture of Tibetan capillaris extract of the present invention in microporous resin;
FIG. 2 is a reversed-phase preparative liquid chromatography separation chart of target component Fr1 of Tibetan capillaris disclosed by the invention;
FIG. 3 is a reversed-phase preparative liquid chromatography separation chart of target component Fr1-1 of Tibetan capillaris disclosed in the present invention;
FIG. 4 is a chromatogram for verifying the purity of pyrone compound Fr1-1-1 (Swermussina) of the new Artemisia capillaris;
FIG. 5 is a low resolution mass spectrum of the pyrone compound SwermussinnA of the Tibetan capillaris;
FIG. 6 shows SwermussinnA, a pyrone compound of the Tibetan capillary artemisia 1 HNMR nuclear magnetic map;
FIG. 7 shows SwermussinnA, a pyrone compound of the Tibetan capillary artemisia 13 A CNMR nuclear magnetic map;
FIG. 8 is a two-dimensional nuclear magnetic diagram of HSQC of the new pyrone compound SwermussinnA of the Tibetan capillaris;
FIG. 9 is an HMBC two-dimensional nuclear magnetic map of the pyrone compound SwermussinnA of the Tibetan capillaris;
FIG. 10 is a two-dimensional nuclear magnetic diagram of H-HCOSY of the new pyrone compound SwermussinnA of the Tibetan capillaris;
FIG. 11 is a NOESY two-dimensional nuclear magnetic diagram of the pyrone compound Swermussina of the Tibetan capillaris of the present invention;
FIG. 12 is a structural diagram of a pyrone compound SwermussinnA of the Tibetan capillaris.
Detailed Description
Example 1, a process for separating and preparing a novel pyrone compound SwermussinA in artemisia scoparia, specifically comprises the following steps:
step 1, extraction: drying 300.0g of Tibetan capillaris in the shade, coarsely crushing, and mixing the materials according to a material-liquid ratio of 1g: extracting with 20mL of methanol at room temperature for 2 times (8 hr each time), filtering, and mixing filtrates to obtain filtrate A, wherein the filtrate A is obtained by mixing silica gel: amount of swertia tibetana extract =3:1, dressing and drying under reduced pressure, wherein the drying under reduced pressure is performed under the following conditions: vacuum degree of 100mbar, temperature of 40 deg.C to obtain sample mixture of Tibetan herba Artemisiae Scopariae extract 35.1g;
step 2, carrying out medium-pressure chromatography rough separation on the microporous resin column: mixing the sample with herba Artemisiae Scopariae extract, separating by medium pressure chromatography with microporous resin, detecting with ultraviolet detector with detection wavelength of 210nm, collecting the first main chromatographic peak fraction in the chromatogram, and drying under reduced pressure to obtain target component 3.9g (see figure 1 for details), wherein the drying under reduced pressure is performed under the following conditions: vacuum degree of 100mbar and temperature of 40 ℃; wherein, the medium pressure chromatographic separation working parameters of the microporous resin column are as follows: 460mm in length and 49mm in diameter of a chromatographic column, CHP20P as a microporous resin column stationary phase, water as a mobile phase A, methanol as a B and dichloromethane as a C, 0 to 150min,0 to 100% of B,150 to 300min,0 to 100% of C, 35g of sample volume and 57mL/min of flow rate;
step 3, preparing a reversed-phase preparation column: dissolving Tibetan herba Artemisiae Scopariae target component Fr1 with 70% methanol, preparing sample with concentration of 50.0mg/mL, filtering with 0.45 μm microporous membrane to obtain target component-containing solution of Tibetan herba Artemisiae Scopariae, i.e. filtrate B, separating the obtained filtrate B with C18 reversed phase preparation column, detecting with ultraviolet detector with detection wavelength of 210nm, collecting corresponding first chromatographic peak fraction Fr1-1 in the preparation chromatogram, and drying under reduced pressure to obtain target component 0.11g (see figure 2 for details); wherein the working parameters of the reversed-phase preparation column preparation are as follows: the preparation method comprises the steps of preparing a column with the length of 250mm and the diameter of 20mm, using a C18 reversed phase chromatographic column with the stationary phase of 5 μm, using a mobile phase A of 0.1% trifluoroacetic acid-water solution and using a mobile phase B of a methanol solution, eluting according to the concentration of 0-30min, using a column B of 8% and a sample injection volume of 0.5mL, and using a flow rate of 19mL/min.
And 4, reversed-phase preparative column purification: dissolving Tibetan capillary artemisia target component Fr1-1 in methanol, preparing a sample with the concentration of 50.0mg/mL, filtering the solution through a 0.45-micrometer microporous filter membrane to obtain a solution containing the target component of Tibetan capillary artemisia, namely a filtrate C, separating the filtrate C through a C18 (w) reverse phase preparation column, detecting the filtrate C through an ultraviolet detector with the detection wavelength of 210nm, collecting corresponding first chromatographic peak fraction Fr1-1-1 in a preparation chromatogram, drying the chromatographic peak fraction Fr1-1-1 under reduced pressure to obtain 3.96mg of a new pyrone compound Fr1-1-1 (detailed in a figure 3) with the purity of more than 95 percent, and naming the pyrone compound Fr1-1-1 (detailed in a figure 12); wherein the working parameters for the preparation of the reversed-phase preparation column are as follows: the preparation method comprises the steps of preparing a column with the length of 250mm and the diameter of 20mm, using a C18 reversed phase chromatographic column with the stationary phase of 5 μm, using a mobile phase A of 0.1% trifluoroacetic acid-water solution and a mobile phase B of a methanol solution, eluting according to the concentration of 11% B within 0-30min, wherein the sample injection volume is 0.5mL, and the flow rate is 19mL/min.
Example 2, step 1, extraction: 1kg of Tibetan virgate wormwood herb is dried in the shade, coarsely crushed and mixed according to the material-liquid ratio of 1g: extracting with 50mL of methanol at room temperature for 3 times (7 h each time), filtering, and mixing filtrates to obtain filtrate A, wherein the filtrate A is obtained by mixing the following components in an amount of silica gel: amount of swertia tibetana extract =3:1, dressing and drying under reduced pressure, wherein the drying under reduced pressure is performed under the following conditions: vacuum degree of 150mbar, temperature of 50 deg.C to obtain sample 117g of Tibetan herba Artemisiae Scopariae extract;
step 2, carrying out medium-pressure chromatography rough separation on the microporous resin column: mixing a sample of the Tibetan capillaris extract, carrying out medium-pressure chromatographic separation on the sample by using microporous resin, detecting the sample by using an ultraviolet detector with the detection wavelength of 210nm, collecting a first main chromatographic peak fraction in a preparative chromatogram, and drying the fraction under reduced pressure to obtain 13g of a target component, wherein the conditions of reduced pressure drying are as follows: vacuum degree of 150mbar and temperature of 50 ℃; wherein, the medium pressure chromatographic separation working parameters of the microporous resin column are as follows: the length of a chromatographic column is 460mm, the diameter is 36mm, the fixed phase of a microporous resin column is CHP20P, the mobile phase A is water, B is methanol, C is dichloromethane, the chromatographic condition is 0 to 150min,0 to 100 percent of B,150 to 300min,0 to 100 percent of C, the sample input is 22g, and the flow rate is 30mL/min;
step 3, preparing a reversed-phase preparation column: dissolving Tibetan herba Artemisiae Scopariae target component Fr1 with 80% methanol, preparing sample with concentration of 70.0mg/mL, filtering with 0.45 μm microporous membrane to obtain target component-containing solution of Tibetan herba Artemisiae Scopariae, i.e. filtrate B, separating the obtained filtrate B with C18 reversed phase preparation column, detecting with ultraviolet detector with detection wavelength of 210nm, collecting corresponding first chromatographic peak fraction Fr1-1 in the preparation chromatogram, and drying chromatographic peak fraction Fr1-1 under reduced pressure to obtain target component 0.37g; wherein the working parameters for the preparation of the reversed-phase preparation column are as follows: preparation of a column having a length of 250mm and a diameter of 20mm, a stationary phase of a C18 reversed phase chromatography column of 5 μm, a mobile phase A of a 0.1% trifluoroacetic acid-water solution, a mobile phase B of a methanol solution, and elution according to the proportion of 0 to 30min,8% B, wherein the sample injection volume is 0.8mL, and the flow rate is 19mL/min.
And 4, reversed-phase preparative column purification: adding methanol into a target component Fr1-1 of Tibetan capillary artemisia for dissolving, preparing a sample with the concentration of 70.0mg/mL, filtering the sample through a 0.45-micron microporous filter membrane to obtain a solution containing the target component of Tibetan capillary artemisia, namely a filtrate C, separating the filtrate C through a C18 (w) reverse phase preparation column, detecting the filtrate C through an ultraviolet detector with the detection wavelength of 210nm, collecting a corresponding first chromatographic peak fraction Fr1-1-1 in a preparation chromatogram, and drying the chromatographic peak fraction Fr1-1-1 under reduced pressure to obtain 13.3mg of a new pyrone compound Fr1-1-1 with the purity of more than 95 percent, wherein the new pyrone compound Fr1-1-1 is named as Swermussina; wherein the working parameters for the preparation of the reversed-phase preparation column are as follows: preparation of a column having a length of 250mm and a diameter of 20mm, a stationary phase of a C18 reversed phase chromatography column of 5 μm, a mobile phase A of a 0.1% trifluoroacetic acid-water solution, a mobile phase B of a methanol solution, and elution according to the proportion of 0 to 30min,11% B, wherein the sample injection volume is 0.8mL, and the flow rate is 19mL/min.
Example 3, a process for the isolation and preparation of a novel pyrone SwermussinA free radical inhibitor from artemisia capillaris thunb, which comprises the following steps:
step 1, extraction: drying 100g of Tibetan capillaris in the shade, coarsely crushing, and mixing the materials according to a material-liquid ratio of 1g: extracting with 5mL of methanol at room temperature for 4 times (6 hr each time), filtering, and mixing filtrates to obtain filtrate A, wherein the filtrate A is obtained by mixing silica gel: amount of swertia tibetana extract =3:1, dressing and drying under reduced pressure, wherein the drying under reduced pressure is performed under the following conditions: vacuum degree of 200mbar, temperature of 60 deg.C to obtain 11.7g of sample mixture of Tibetan herba Artemisiae Scopariae extract;
step 2, carrying out medium-pressure chromatography rough separation on the microporous resin column: mixing a sample of the Tibetan capillaris extract, carrying out medium-pressure chromatographic separation on the sample by using microporous resin, detecting the sample by using an ultraviolet detector with the detection wavelength of 210nm, collecting a first main chromatographic peak fraction in a preparative chromatogram, and drying the fraction under reduced pressure to obtain 1.3g of a target component, wherein the conditions of reduced pressure drying are as follows: vacuum degree of 200mbar and temperature of 60 ℃; wherein, the medium-pressure chromatographic separation working parameters of the microporous resin column are as follows: the length of a chromatographic column is 460mm, the diameter is 15mm, the stationary phase of a microporous resin column is CHP20P, the mobile phase A is water, B is methanol, C is dichloromethane, the chromatographic conditions are 0 to 150min,0 to 100 percent of B,150 to 300min,0 to 100 percent of C, the sample introduction amount is 15g, and the flow rate is 8mL/min;
step 3, preparing a reversed-phase preparation column: dissolving Tibetan capillary wormwood target component Fr1 in 90% methanol, preparing a sample with a concentration of 90.0mg/mL, filtering with a 0.45 μm microporous membrane to obtain a solution containing the target component, namely filtrate B, separating the obtained filtrate B with a C18 reversed phase preparation column, detecting with an ultraviolet detector with a detection wavelength of 210nm, collecting corresponding first chromatographic peak fraction Fr1-1 in a preparation chromatogram, and drying under reduced pressure to obtain target component Fr1 mg; wherein the working parameters of the reversed-phase preparation column preparation are as follows: the preparation method comprises the steps of preparing a column with the length of 250mm and the diameter of 20mm, using a C18 reversed phase chromatographic column with the stationary phase of 5 μm, using a mobile phase A of 0.1% trifluoroacetic acid-water solution and a mobile phase B of a methanol solution, eluting according to the concentration of 0-30min, using the concentration of 8% B, wherein the sample injection volume is 0.2mL, and the flow rate is 19mL/min.
And 4, reversed-phase preparative column purification: dissolving Tibetan capillary artemisia target component Fr1-1 in methanol, preparing a sample with the concentration of 90.0mg/mL, filtering the solution through a 0.45-micrometer microporous filter membrane to obtain a solution containing the target component of Tibetan capillary artemisia, namely a filtrate C, separating the filtrate C through a C18 (w) reverse phase preparation column, detecting the filtrate C through an ultraviolet detector with the detection wavelength of 210nm, collecting corresponding first chromatographic peak fraction Fr1-1-1 in a preparation chromatogram, and drying the chromatographic peak fraction Fr1-1-1 under reduced pressure to obtain 1.32mg of a new pyrone compound Fr1-1-1 with the purity of more than 95 percent, wherein the new pyrone compound Fr1-1-1 is named as Swermussina; wherein the working parameters for the preparation of the reversed-phase preparation column are as follows: the preparation method comprises the steps of preparing a column with the length of 250mm and the diameter of 20mm, using a C18 reversed phase chromatographic column with the stationary phase of 5 μm, using a mobile phase A of 0.1% trifluoroacetic acid-water solution and a mobile phase B of a methanol solution, eluting according to the concentration of 11% B within 0-30min, wherein the sample injection volume is 0.2mL, and the flow rate is 19mL/min.
The nuclear magnetic signal assignments for the furanone compound prepared in the three examples above are:
ESI-MSm/z:229.12[M-H] - , 1 HNMR(600MHz,CD 3 OD):δ4.49(1H,d,J=12.6Hz,3-Ha),4.51(1H,d,J=12.6Hz,3-Hb),2.85(1H,brd,J=18.9Hz,6-Ha),2.96(1H,ddd,J=18.9,12.2,6.0Hz,6-Hb),4.45(1H,ddd,J=11.0,6.0,2.1Hz,7-Ha),4.84(1H,ddd,J=12.2,11.0,3.3Hz,7-Hb),2.30,2.41(each1H,m,8-H),1.05(3H,t,J=7.5Hz,10-H)。 13 CNMR(151MHz,CD 3 OD): δ170.0(C-11),165.5(C-1),146.1(C-5),132.9(C-9),72.8(C-3),67.2(C-4),66.4(C-7),26.8(C-6),21.6(C-8),12.5(C-10)。
the mass spectrum and nuclear magnetic signal diagram of the pyrone compound SwermussinnA are shown in figures 5-11.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (9)

1. A method for separating and preparing a novel pyrone compound in Tibetan capillary is characterized by comprising the following steps:
step 1, extraction: drying Tibetan herba Artemisiae Scopariae in the shade, pulverizing, extracting with methanol, filtering, mixing filtrates to obtain filtrate A, mixing filtrate A with silica gel, and drying under reduced pressure to obtain Tibetan herba Artemisiae Scopariae extract sample;
step 2, carrying out medium-pressure chromatography rough separation on the microporous resin column: mixing a sample with the Tibetan capillaris extract, separating the sample by medium-pressure chromatography filled with microporous resin, detecting by an ultraviolet detector, collecting a first main chromatographic peak fraction in a preparative chromatogram, and drying the fraction under reduced pressure to obtain a crude isolate Fr1 containing a target component;
step 3, preparing a reversed-phase preparation column: adding methanol with volume concentration of 70-90% into the crude isolate Fr1 to dissolve to prepare a sample solution, filtering by a 0.45 mu m microporous filter membrane to obtain a filtrate B containing a target component, separating the obtained filtrate B by a C18 reverse phase preparation column, detecting by an ultraviolet detector, collecting a corresponding first chromatographic peak fraction in a preparation chromatogram, and drying under reduced pressure to obtain a crude isolate Fr1-1 containing the target component;
and 4, reversed-phase preparative column purification: and (2) adding methanol into the crude isolate Fr1-1 to dissolve to prepare a sample solution, filtering by a 0.45-micrometer microporous filter membrane to obtain a filtrate C containing a target component, separating the filtrate C by a C18 reversed-phase preparation column, detecting by an ultraviolet detector, collecting a corresponding first chromatographic peak fraction in a preparative chromatogram, and drying under reduced pressure to obtain the isolate Fr1-1-1, namely the new pyrone compound.
2. The method for separating and preparing novel pyrones from swertia pseudochinensis benth as claimed in claim 1, wherein the methanol extraction in step 1 is performed at a ratio of 1g:5 to 100mL, and 2~4 times of extraction at room temperature for 6 to 8h each time.
3. The method for separating and preparing pyrones from swertia pseudochinensis Benth in claim 2, wherein the mass ratio of silica gel to swertia pseudochinensis Benth extract in step 1 is 3:1.
4. The method according to claim 3, wherein the crude isolate Fr1 is added with methanol to prepare a sample solution with a concentration of 50.0 to 100.0mg/mL in step 3.
5. The method for separating and preparing a pyrone compound from Tibetan capillary artemisia as claimed in claim 4, wherein in step 4, methanol is added to the crude isolate Fr1-1 to prepare a sample solution with a concentration of 50.0 to 100.0mg/mL.
6. The method for separating and preparing pyrones from Tibetan capillary artemisia as claimed in claim 5, wherein the drying under reduced pressure in steps 1-4 is performed under the following conditions: the vacuum degree is 50 to 250mbar, and the temperature is 40 to 60 ℃; the detection wavelength of the ultraviolet detector is 210nm.
7. The method for separating and preparing pyrones from plant of claim 6, wherein in step 2, the medium-pressure chromatographic column has the following specifications: the length of the column is 460mm, the diameter is 15 to 49mm, and the stationary phase of the microporous resin is CHP20P; the mobile phase A is water, the mobile phase B is methanol, the mobile phase C is dichloromethane, the chromatographic conditions are 0 to 150min,0 to 100 percent of B,150 to 300min,0 to 100 percent of C, the sample volume is 5 to 50g, and the flow rate is 8 to 57mL/min.
8. The method for separating and preparing a pyrone compound from swertia pseudochinensis benth as claimed in claim 7, wherein in step 3, the C18 reverse phase preparation column has the following specifications: the column length is 250mm, the diameter is 20mm, and the granularity is 5 mu m; the mobile phase A is 0.1% trifluoroacetic acid-water solution, the mobile phase B is methanol solution, elution is carried out by 8%B under the conditions of 0 to 30min, the sample injection volume is 0.2 to 1.0mL, and the flow rate is 19mL/min.
9. The method of claim 8, wherein in step 4, the C18 reverse phase preparative column has the following specifications: the column length was 250mm, the diameter was 20mm, the particle size was 5 μm, the mobile phase A was a 0.1% trifluoroacetic acid-water solution, the mobile phase B was a methanol solution, and the elution was carried out at 0 to 30min,11% B, the injection volume was 0.2 to 1.0mL, and the flow rate was 19mL/min.
CN202211030057.7A 2022-08-26 2022-08-26 Method for separating and preparing pyrone compound in Tibetan capillary Pending CN115385928A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103408615A (en) * 2013-07-22 2013-11-27 中国科学院西北高原生物研究所 Preparation method for chemical reference substance of sweroside in Tibetan capillary artemisia medicinal material

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103408615A (en) * 2013-07-22 2013-11-27 中国科学院西北高原生物研究所 Preparation method for chemical reference substance of sweroside in Tibetan capillary artemisia medicinal material

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Title
杨帆: "藏茵陈的化学成分研究", 《成都师范学院学报》 *
王世盛: "藏茵陈活性组分的制备分离和化学表征", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 *

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