CN115368359A - Alkaloid antioxidant and preparation method and application thereof - Google Patents
Alkaloid antioxidant and preparation method and application thereof Download PDFInfo
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- CN115368359A CN115368359A CN202211120948.1A CN202211120948A CN115368359A CN 115368359 A CN115368359 A CN 115368359A CN 202211120948 A CN202211120948 A CN 202211120948A CN 115368359 A CN115368359 A CN 115368359A
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- 230000003078 antioxidant effect Effects 0.000 title claims abstract description 34
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- 238000002360 preparation method Methods 0.000 title claims abstract description 27
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- OEKUVLQNKPXSOY-UHFFFAOYSA-N quercetin 3-O-beta-D-glucopyranosyl(1->3)-alpha-L-rhamnopyranosyl(1->6)-beta-d-galactopyranoside Natural products OC1C(O)C(C(O)C)OC1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OEKUVLQNKPXSOY-UHFFFAOYSA-N 0.000 description 1
- QPHXPNUXTNHJOF-UHFFFAOYSA-N quercetin-7-O-beta-L-rhamnopyranoside Natural products OC1C(O)C(O)C(C)OC1OC1=CC(O)=C2C(=O)C(O)=C(C=3C=C(O)C(O)=CC=3)OC2=C1 QPHXPNUXTNHJOF-UHFFFAOYSA-N 0.000 description 1
- OXGUCUVFOIWWQJ-HQBVPOQASA-N quercitrin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OXGUCUVFOIWWQJ-HQBVPOQASA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/04—Ortho-condensed systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
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- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
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- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an alkaloid antioxidant, a preparation method and application thereof, and belongs to the technical field of extraction of alkaloid compounds. The preparation method comprises the following steps: heating and refluxing the whole herb of the Lepidium europaeum with ethanol to obtain a crude extract; and then performing silica gel column chromatography, reversed-phase high-pressure liquid preparative chromatographic separation and reversed-phase preparative column purification to obtain the alkaloid antioxidant. The alkaloid antioxidant prepared by separation has obvious total antioxidant capacity. In addition, the sources of the raw material of the Lepidium europaeum are wide, the methods such as ethanol heating reflux extraction and the like can realize large-scale operation, and the high-pressure liquid phase preparative chromatography can ensure that the purity of the product is more than 98 percent.
Description
Technical Field
The invention belongs to the technical field of alkaloid compound extraction, and particularly relates to an alkaloid antioxidant and a preparation method and application thereof.
Background
Oxidative Stress (OS), a condition of imbalance between Oxidative and antioxidant effects in the body, is prone to oxidation, leading to inflammatory infiltration of neutrophils, increased secretion of proteases, and production of a number of Oxidative intermediates. Oxidative stress is a negative effect produced in the body by free radicals and is considered to be an important factor in aging and diseases.
Lepidium latifolium L, which is a Lepidium plant of the family Brassicaceae (Brassicaceae), is also called Da La, la Rou Dou, zhi Li Cao. The wide-leaf Lepidium unibracteatum is originated from the field, the ridge, the ditch and the valley, is distributed in Gansu, qinghai, ningxia and other places, is widely distributed, has rich resources and has certain development and utilization values. According to the records of medicinal plants in the desert area of China, the Lepidium europaeum has the effects of clearing heat and drying dampness, and treating bacillary dysentery and enteritis. Modern pharmaceutical research shows that the Lepidium europaeum has certain antioxidant and anti-aging effects, so that the method for separating and identifying the antioxidant from the Lepidium europaeum has important application value in the aspects of antioxidant, anti-aging and related medicine synthesis.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an alkaloid antioxidant and a preparation method and application thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention adopts one of the technical schemes: an alkaloid antioxidant with molecular formula of C 16 H 12 N 2 O 2 The structural formula is as follows:
the second technical scheme of the invention is as follows: the preparation method of the alkaloid antioxidant comprises the following steps:
(1) Extracting herba Lespedezae Buergeri with ethanol under reflux, and concentrating the extractive solution to obtain crude extract;
(2) Sequentially extracting the crude extract obtained in the step (1) by using petroleum ether, ethyl acetate and n-butyl alcohol, and concentrating the extract to obtain a petroleum ether part, an ethyl acetate part and an n-butyl alcohol part;
(3) Separating the n-butanol part obtained in the step (2) by using a normal phase silica gel column chromatography, performing gradient elution by using a dichloromethane-methanol mixed mobile phase, and concentrating and drying the eluent to obtain components Fr.1-Fr.4 in sequence;
(4) Separating the component Fr.4 obtained in the step (3) by using microporous resin open column chromatography, performing gradient elution by using a methanol-water mixed mobile phase, and concentrating the eluent to sequentially obtain components Fr.4-1-Fr.4-10;
(5) Separating the component Fr.4-2 obtained in the step (4) by using a reversed-phase high-pressure liquid chromatography column, eluting by using a formic acid solution-acetonitrile mixed mobile phase, detecting an eluent by using an ultraviolet detector, collecting chromatographic peak fractions, and drying to obtain a component Fr.4-2-1;
(6) And (3) purifying the component Fr.4-2-1 obtained in the step (5) by using a reverse phase preparation column, eluting by using a formic acid solution-acetonitrile mixed mobile phase, detecting eluent by using an ultraviolet detector, collecting chromatographic peak fractions, and drying to obtain a component Fr.4-2-1-2, namely the alkaloid antioxidant.
Further, in the step (1), the Lepidium europaeum is Lepidium wide leaf powder, and the specific preparation method is that whole grass of the Lepidium wide leaf is dried in the shade, crushed and sieved by a 40-mesh sieve.
Further, in the step (1), the volume fraction of the ethanol is 95%, and the feed-liquid ratio of the cress broadleaf herb to the ethanol is 1kg: (5-50) L; the temperature of the reflux extraction is 70 ℃, the time is 2-4 h, and the times are 2-4; the vacuum degree of the concentration is 0.07-0.09 MPa, and the temperature is 50-60 ℃.
Further, in the step (2), the volume ratio of the crude extract to the petroleum ether, ethyl acetate or n-butanol is 1:1; the extraction times are 2-4; the vacuum degree of the concentration is 0.07-0.09 MPa, and the temperature is 50-60 ℃.
Further, in the step (3), before the normal phase silica gel column chromatography, the n-butanol part needs to be mixed with 200-300 mesh silica gel, and the material-liquid ratio of the n-butanol part to the silica gel is 1g: 1-3 mL; the volume ratio of the dichloromethane to the methanol in the dichloromethane-methanol mixed mobile phase gradient elution is 15: 1. 10: 1. 5: 1. 1:1; the vacuum degree of the drying is 0.07-0.09 MPa, and the temperature is 50-60 ℃.
Further, in the step (4), the stationary phase of the microporous resin open column chromatography is MCI microporous resin; before the separation by using the microporous resin open column chromatography, a component Fr.4 is dissolved in water and passes through a 0.45-micron microporous filter membrane, wherein the mass-to-volume ratio of Fr.4 to water is 1:1; the volume ratio of methanol in the gradient elution of the methanol-water mixed mobile phase is 0, 10%,20%,30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% in sequence; the vacuum degree of the concentration is 0.07-0.09 MPa, and the temperature is 50-60 ℃.
Further, in the step (5), the length of the reversed phase high pressure liquid chromatography column is 250mm, the diameter is 20mm, and the stationary phase is Dubhe C18 with the diameter of 5 μm; the volume fraction of acetonitrile in the formic acid solution-acetonitrile mixed mobile phase is 14%, and the volume fraction of formic acid in the formic acid solution is 0.2%; the elution time is 55min, and the elution flow rate is 19mL/min; the wavelength of the ultraviolet detector is 254nm; the vacuum degree of the drying is 0.07-0.09 MPa, and the temperature is 50-60 ℃.
Further, in the step (6), the reversed phase preparation column has a length of 250mm, a diameter of 10mm and a stationary phase of 5 μm for a Unitry analytical column; the volume fraction of acetonitrile in the formic acid solution-acetonitrile mixed mobile phase is 5%, and the volume fraction of formic acid in the formic acid solution is 0.2%; the elution time is 60min, and the elution flow rate is 5.0mL/min; the wavelength of the ultraviolet detector is 254nm; the vacuum degree of the drying is 0.07-0.09 MPa, and the temperature is 50-60 ℃.
The third technical scheme of the invention is as follows: the alkaloid antioxidant is applied to the preparation of antioxidant drugs.
Compared with the prior art, the invention has the following beneficial effects:
(1) The alkaloid antioxidant prepared by separation has obvious total antioxidant capacity;
(2) The purity of the alkaloid antioxidant prepared by separation can reach 98 percent;
(3) The raw material of the Lepidium europaeum has wide sources, and the methods of extraction, silica gel column chromatography, high pressure liquid chromatography and the like can all realize large-scale operation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the preparation of the alkaloid antioxidants prepared in example 1 of the present invention 1 H-NMR spectrum;
FIG. 2 shows the preparation of the alkaloid antioxidants of example 1 of the present invention 13 C-NMR spectrum;
fig. 3 is a graph showing the antioxidant activity of the alkaloid antioxidants, the vitamin C solution and the quercetin solution prepared in example 1 of the present invention.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. It is intended that the specification and examples be considered as exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
Separation and preparation of alkaloid antioxidant in Lepidium europaeum
(1) Pretreatment of raw materials: drying the whole herb of the Lepidium europaeum in the shade, crushing, and sieving with a 40-mesh sieve to obtain raw material powder;
(2) Extraction: extracting 5kg of the raw material powder obtained in the step (1) with 50L 95vol.% ethanol under reflux at 70 deg.C for 4h, repeating the extraction for 4 times, mixing the extractive solutions, and concentrating under reduced pressure at 60 deg.C under 0.09MPa to obtain crude extract;
(3) And (3) extraction: diluting the crude extract obtained in step (2) with water to 4L, sequentially extracting with 4L petroleum ether, ethyl acetate and n-butanol, extracting with each extractant for 4 times to obtain petroleum ether fraction, ethyl acetate fraction and n-butanol fraction, and concentrating each fraction under reduced pressure at vacuum degree of 0.09MPa and temperature of 60 deg.C;
(4) Roughly separating by silica gel column chromatography: mixing 0.2L of n-butanol part obtained in the step (3) with 200g of 200-300 mesh silica gel, loading on a normal phase silica gel column, and mixing the mixture with a solvent with a volume ratio of 15: 1. 10: 1. 5: 1. 1:1, performing gradient elution on the dichloromethane-methanol mixed mobile phase, collecting eluent obtained by elution of the dichloromethane-methanol mixed mobile phase with the volume ratio of 1:1, performing reduced pressure concentration on the eluent under the conditions that the vacuum degree is 0.09MPa and the temperature is 60 ℃, and drying in an oven at 50 ℃ to obtain a component Fr.4;
(5) Roughly separating gel column chromatography: dissolving 10g of the component Fr.4 obtained in the step (4) in 10mL of water, filtering the solution through a 0.45-micron microporous membrane, separating the solution by utilizing a microporous resin open column chromatography, wherein the stationary phase of the microporous resin open column chromatography is MCI microporous resin, the mobile phase A is water, and the mobile phase B is methanol, wherein the volume concentration of the methanol is linearly increased from 0, 10 percent, 20 percent, 30 percent, …,100 percent, and performing gradient elution to respectively obtain 10 kinds of eluents in sections, and performing reduced pressure concentration on the 10 kinds of eluents under the conditions that the vacuum degree is 0.09MPa and the temperature is 60 ℃ to obtain the components Fr.4-1-Fr.4-10;
(6) High-pressure liquid chromatography separation: separating 2mL of the component Fr.4-2 obtained in the step (5) by using a reversed-phase high-pressure liquid chromatography, wherein the length of a column of the reversed-phase high-pressure liquid chromatography is 250mm, the diameter of the column is 20mm, the stationary phase is Dubhe C18 with the diameter of 5 microns, the mobile phase A is 0.2% formic acid-water, the mobile phase B is acetonitrile, the volume fraction of the acetonitrile is 14%, the sample injection amount is 0.5mL, the flow rate of the mobile phase is 19mL/min, the elution time is 55min, detecting the eluent by using an ultraviolet detector with the detection wavelength of 254nm, and injecting samples for 4 times under the same chromatographic condition. Collecting and combining chromatographic peak fractions in a chromatogram, and drying the fractions under reduced pressure at the vacuum degree of 0.09MPa and the temperature of 60 ℃ to obtain a component Fr.4-2-1;
(7) And (3) reverse preparative column purification: dissolving the component Fr.4-2-1 obtained in the step (6) in 1mL of 10% methanol, purifying the dissolved Fr.4-2-1 by using a reverse preparation column, wherein the length of the reverse preparation column is 250mm, the diameter of the reverse preparation column is 10mm, the stationary phase of the reverse preparation column is a 5 mu m Unitry analytical column, the mobile phase A is 0.2% formic acid-water solution, the mobile phase B is acetonitrile solution, the volume fraction of the acetonitrile is 5%, the sample injection amount is 200 mu L, the flow rate of the mobile phase is 5.0mL/min, the elution time is 60min, detecting the eluent by an ultraviolet detector with the detection wavelength of 254nm, and carrying out sample injection for 5 times under the same chromatographic condition. Collecting and combining chromatographic peak fractions in a chromatogram, and drying the fractions under reduced pressure at the vacuum degree of 0.09MPa and the temperature of 60 ℃ to obtain a component Fr.4-2-1-2, namely the alkaloid antioxidant in the Lepidium europaeum.
The purity of the alkaloid antioxidant prepared in this example was 98%.
Example 2
Structural identification of alkaloid antioxidants
The alkaloid antioxidants prepared in example 1 were subjected to nuclear magnetic resonance ( 1 H-NMR、 13 C-NMR), the results are shown in FIGS. 1-2, in which FIG. 1 is that of the alkaloid antioxidants 1 H-NMR spectrum, FIG. 2 for a alkaloid antioxidant 13 C-NMR spectrum, the specific physicochemical data of the alkaloid antioxidants are as follows:
fr.4-2-1-2 of yellow powder, 1 H NMR(600MHz,Methanol-d4)δ8.30(1H,d,J＝5.2Hz,H-3),8.20(1H,d,J＝7.8Hz,H-5),8.04(1H,d,J＝5.2Hz,H-4),7.72(1H,d,J＝8.2Hz,H-8),7.60(2H,t,J＝7.5Hz,H-7),7.30(1H,t,J＝7.5Hz,H-6),7.24(1H,d,J＝3.2Hz,H-3'),6.60(1H,d,J＝3.2Hz,H-4'),4.77(2H,s,H-6'). 13 C NMR(151MHz,Methanol-d4)δ157.34(C-2'),154.07(C-5'),143.15(C-9b),138.40(C-3),134.23(C-1),132.51(C-9a),132.31(C-4b),130.12(C-4a),122.65(C-5),122.12(C-7),121.28(C-6),115.04(C-8),113.30(C-4),111.18(C-3'),111.06(C-4'),57.58(C-6')。
the structural formula of the obtained alkaloid is as follows:
examples of effects
Experiment of antioxidant Activity
The total antioxidant capacity (T-AOC) determination kit is adopted for the alkaloid antioxidant prepared in the example 1 to determine the total antioxidant capacity of a sample, and the specific steps are as follows:
(1) Preparation of FeSO 4 -7H 2 O standard solution: 27.8mg of FeSO were weighed out 4 -7H 2 O standard (provided by Total Oxidation resistance (T-AOC) assay kit), dissolved in 1mL of 50% methanol to prepare a 100mM solution, diluted to standard solutions of concentrations of 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5mM, respectively;
(2) Preparation of positive control: precisely weighing 3.6mg of vitamin C to prepare a vitamin C solution with the concentration of 3.6mg/mL, adding 50% methanol to dilute to 0.2mg/mL, precisely weighing 6.1mg of quercetin to prepare a quercetin solution with the concentration of 6.1mg/mL, and adding 50% methanol to dilute to 0.2mg/mL; preparing a sample solution: 5.3mg Fr.4-2-1-2Perlolyrin was precisely weighed to prepare a 5.3mg/mL Perlolyrin solution, and diluted to 0.2mg/mL with 50% methanol.
(3) In a 96-well plate, a blank well to which 5. Mu.L of 50% methanol and 180. Mu.L of FRAP working solution were added, a standard well to which FeSO was added, and a measurement well were set 4 -7H 2 Adding 5 mu L of sample solution to be detected and 180 mu L of FRAP working solution into a measuring hole of standard substance solution with different concentrations, fully and uniformly mixing, incubating at 37 ℃ for 3-5min, measuring the light absorption value at 593nm, and repeating each sample for three times; and reading the OD value of each hole by using a microplate reader.
(4) Standard curve: subtracting the light absorption value of the blank hole from each hole, taking the light absorption value of the standard substance as an abscissa, taking the concentration of the standard substance corresponding to each light absorption value as an ordinate to prepare a standard curve, and preparing a curve formula by using EXCEL;
(5) And (3) calculating: substituting the absorbance value (blank absorbance value needs to be subtracted) measured by the sample measuring tube into a standard curve formula, and obtaining a result by FeSO 4 -7H 2 The concentration (mM) of the O standard solution.
The results of the positive control using vitamin C solution and quercetin solution are shown in fig. 3, and it can be found that the total antioxidant capacity is: alkaloid antioxidant (perlolysine, 0.28 mM) > quercetin (Quercitrin, 0.22 mM) > Vitamin C (Vitamin C,0.06 mM).
The above description is only for the preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (10)
1. An alkaloid antioxidant characterized by having a molecular formula of C 16 H 12 N 2 O 2 The structural formula is as follows:
2. a process for the preparation of the alkaloid antioxidants of claim 1, characterized in that it comprises the following steps:
(1) Extracting herba Lespedezae Buergeri with ethanol under reflux, and concentrating the extractive solution to obtain crude extract;
(2) Sequentially extracting the crude extract obtained in the step (1) with petroleum ether, ethyl acetate and n-butanol, and concentrating the extract to obtain a petroleum ether part, an ethyl acetate part and an n-butanol part;
(3) Separating the n-butanol part obtained in the step (2) by using normal phase silica gel column chromatography, performing gradient elution by using a dichloromethane-methanol mixed mobile phase, and concentrating and drying the eluent to obtain components Fr.1-Fr.4 in sequence;
(4) Separating the component Fr.4 obtained in the step (3) by using microporous resin open column chromatography, performing gradient elution by using a methanol-water mixed mobile phase, and concentrating the eluent to sequentially obtain components Fr.4-1-Fr.4-10;
(5) Separating the component Fr.4-2 obtained in the step (4) by using a reversed-phase high-pressure liquid chromatography column, eluting by using a formic acid solution-acetonitrile mixed mobile phase, detecting an eluent by using an ultraviolet detector, collecting chromatographic peak fractions, and drying to obtain a component Fr.4-2-1;
(6) And (3) purifying the component Fr.4-2-1 obtained in the step (5) by using a reverse phase preparation column, eluting by using a formic acid solution-acetonitrile mixed mobile phase, detecting eluent by using an ultraviolet detector, collecting chromatographic peak fractions, and drying to obtain a component Fr.4-2-1-2, namely the alkaloid antioxidant.
3. The preparation method according to claim 2, wherein the Lepidium europaeum in step (1) is Lepidium europaeum powder, and is specifically prepared by drying whole Lepidium europaeum in the shade, pulverizing, and sieving with a 40-mesh sieve.
4. The preparation method according to claim 2, wherein in the step (1), the volume fraction of the ethanol is 95%, and the feed-liquid ratio of the cress to the ethanol is 1kg: (5-50) L; the temperature of the reflux extraction is 70 ℃, the time is 2-4 h, and the times are 2-4; the vacuum degree of the concentration is 0.07-0.09 MPa, and the temperature is 50-60 ℃.
5. The method according to claim 2, wherein in the step (2), the volume ratio of the crude extract to the petroleum ether, ethyl acetate or n-butanol is 1:1; the extraction times are 2-4; the vacuum degree of the concentration is 0.07-0.09 MPa, and the temperature is 50-60 ℃.
6. The preparation method according to claim 2, wherein in the step (3), before the normal phase silica gel column chromatography, the n-butanol part is stirred with 200-300 mesh silica gel, and the feed-to-liquid ratio of the n-butanol part to the silica gel is 1g: 1-3 mL; the volume ratio of the dichloromethane to the methanol in the dichloromethane-methanol mixed mobile phase gradient elution is 15: 1. 10: 1. 5: 1. 1:1; the vacuum degree of the drying is 0.07-0.09 MPa, and the temperature is 50-60 ℃.
7. The preparation method according to claim 2, wherein in the step (4), the stationary phase of the microporous resin open column chromatography is MCI microporous resin; before the separation by using the microporous resin open column chromatography, a component Fr.4 is dissolved in water and passes through a 0.45-micron microporous filter membrane, wherein the mass-to-volume ratio of Fr.4 to water is 1:1; the volume ratio of methanol in the gradient elution of the methanol-water mixed mobile phase is 0, 10%,20%,30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% in sequence; the vacuum degree of the concentration is 0.07-0.09 MPa, and the temperature is 50-60 ℃.
8. The production method according to claim 2, wherein in the step (5), the reversed-phase high-pressure liquid chromatography column has a length of 250mm, a diameter of 20mm, and a stationary phase of 5 μm of Dubhe C18; the volume fraction of acetonitrile in the formic acid solution-acetonitrile mixed mobile phase is 14%, and the volume fraction of formic acid in the formic acid solution is 0.2%; the elution time is 55min, and the elution flow rate is 19mL/min; the wavelength of the ultraviolet detector is 254nm; the vacuum degree of the drying is 0.07-0.09 MPa, and the temperature is 50-60 ℃.
9. The method according to claim 2, wherein in the step (6), the reversed-phase preparative column has a length of 250mm, a diameter of 10mm, and a stationary phase of 5 μm for a Unitry analytical column; the volume fraction of acetonitrile in the formic acid solution-acetonitrile mixed mobile phase is 5%, and the volume fraction of formic acid in the formic acid solution is 0.2%; the elution time is 60min, and the elution flow rate is 5.0mL/min; the wavelength of the ultraviolet detector is 254nm; the vacuum degree of the drying is 0.07-0.09 MPa, and the temperature is 50-60 ℃.
10. Use of the alkaloid antioxidants of claim 1 for the preparation of anti-oxidant medicaments.
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