Anion exchange magnetic bead for extracting radix pseudostellariae cyclic peptide and application method of anion exchange magnetic bead
By developing anion exchange magnetic beads for extracting pyrzolid peptides, the problem of difficult to efficiently isolate and purify pyrzolid peptides in the prior art is solved, and an efficient, selective and reusable polypeptide extraction effect is achieved.
Patent Information
- Application Number
- CN202411926588.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-25
- Publication Date
- 2025-03-28
AI Technical Summary
It is difficult to efficiently isolate and purify pyrogenic peptides, especially cyclic peptides with molecular weight less than 1500 Da. Traditional methods have problems such as risk of loss, low resolution, and complex processing.
Anion exchange magnetic beads for extracting genus gin peptides were developed. Anion exchange magnetic beads with selective adsorption ability were formed by adding olefin-based magnetic beads, monomers, crosslinking agents and initiators to the reaction system, and after stirring, heating and rapid heating treatment.
It has achieved efficient and selective isolation and purification of pyrogenic ginseng peptide, with high recovery rate, high efficiency, reusable, wide applicability, low cost and environmentally friendly.
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Figure CN119702093A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of anion exchange magnetic beads, and in particular to anion exchange magnetic beads for extracting Pseudostellaria heterophylla cyclic peptide and an application method thereof. Background Art
[0002] Pseudostellariae Radix is the dried root of the perennial herbaceous plant Pseudostellariae Radix. It is one of the traditional Chinese tonic herbs with both medicinal and therapeutic effects. It is mainly distributed in Fujian, Guizhou, Anhui, Jiangsu and Shandong. Pseudostellariae Radix has the effects of invigorating qi and strengthening the spleen, promoting body fluid and moistening the lungs. At present, Chinese patent medicines for digestion and strengthening the spleen with Pseudostellariae Radix as the main drug, such as Jianwei Xiaoshi Tablets and Compound Pseudostellariae Radix Granules, are used in clinical practice; in terms of dietary health care, Pseudostellariae Radix can be used for stewing soup, soaking wine and drinking tea, etc., which can strengthen the spleen and replenish qi and is a tonic suitable for all ages. Previous studies have shown that Pseudostellariae Radix contains chemical components such as sugars, glycosides, cyclic peptides, alkaloids and phenols, which have pharmacological effects such as hypoglycemic, anti-inflammatory, anti-cancer and immunomodulatory. Among them, cyclopeptides are commonly found in Caryophyllaceae plants. They are a class of cyclic compounds formed by amino acids connected by peptide bonds. Up to now, there are fourteen cyclopeptides isolated from Pseudostellariae with detailed spectral data. Pseudostellariae cyclopeptide B is the main component. Pharmacological studies have found that Pseudostellariae cyclopeptide B exerts anti-inflammatory, anti-tumor and cell protective effects, and has broad clinical application prospects.
[0003] my country has very rich resources of Radix Pseudostellariae Radix. Separation of Radix Pseudostellariae Radix Cyclotide has high clinical application value. Common methods for separating polypeptides include: dialysis purification, gel filtration, ultrafiltration, solid phase extraction, etc. These separation methods have their own defects. When the molecular weight of the polypeptide is greater than 3000Da, a dialysis bag with a filter limit of about 1500Da can be selected. Since the filter limit is too close to the molecular weight of the product, there is a risk of loss. If the filter limit is too small than the molecular weight, the purification purpose cannot be achieved. The mechanism of gel filtration is the molecular sieve effect, which is separated and purified according to the size of the molecular weight, but the resolution is very low, and it is difficult to separate impurities with similar molecular weights; ultrafiltration is a pressurized membrane separation technology, which is widely used in the separation and purification of various biomacromolecules containing small molecular solutes, but due to the influence of concentration polarization, membrane pore blockage and other factors, the membrane flux decreases with the extension of the running time. Radix Pseudostellariae Radix Cyclotide with a molecular weight less than 1500Da is not suitable for the above separation methods. Simple solid phase extraction can use C18 filter layer and ion exchange filter layer, with high product recovery rate, reusable filter layer and low cost. However, this chromatography method has the limitations of complicated processing process, long time consumption and low degree of automation.
[0004] Magnetic nanomaterials are widely used in the fields of environment, medicine, catalysis and energy due to their good stability, rapid separation ability and easy modification of surface functional groups. At present, the main matrices of magnetic nanomaterials on the market can be divided into silicon-based, polymer-based and agarose-based. The magnetic solid phase extraction technology based on magnetic nanomaterials can combine the advantages of the above-mentioned separation methods. It can not only eliminate the interference of a large number of highly polar small molecules and weakly polar lipids, but also quickly separate impurities with similar molecular weight, electrical properties and hydrophobicity from the target cyclic peptide, and has almost no damage to the activity of the cyclic peptide. In addition, this technology combined with automated equipment can achieve high-throughput extraction of Pseudostellaria heterophylla cyclic peptides, which has good market prospects.
[0005] Therefore, it is necessary to develop an anion exchange magnetic bead for extracting Pseudo-P ... Summary of the invention
[0006] In order to overcome the deficiencies of the prior art, the first object of the present invention is to provide an anion exchange magnetic bead for extracting Pseudostellaria baicalensis cyclic peptide, wherein the anion exchange magnetic bead is prepared as follows:
[0007] Step 1: adding olefin-based magnetic beads, monomers, cross-linking agents and initiators to the reaction system, stirring and heating; wherein the volume percentage of the olefin-based magnetic beads in the reaction system is 0.1-0.5%, the volume percentage of the monomers in the reaction system is 0.5-5%, the volume percentage of the cross-linking agent in the reaction system is 0.5-5%, and the volume percentage of the initiator in the reaction system is 0.1-1%;
[0008] Step 2: Rapidly heat the reaction system to 70-80°C and reflux at a constant temperature;
[0009] Step 3: After the reaction is completed, wash with a solvent.
[0010] Preferably, the olefin-based magnetic beads are at least one of silicon-based olefin magnetic beads, polymer olefin magnetic microspheres, and wrinkled porous polymer olefin magnetic microspheres.
[0011] Preferably, the monomer is at least one of 4-aminostyrene, 4-dimethylaminostyrene, N-vinylpyrrolidone, N,N-dimethylallylamine, benzylvinyltrimethylammonium chloride, allyltrimethylammonium chloride, (3-acrylamidopropyl)trimethylammonium chloride, dimethylaminopropylacrylamide, 1-vinylimidazole, 1-allylimidazole, 1-allyl-3-methylimidazole chloride, N,N,N-trimethyl-(1-(4-vinylphenyl))methylammonium chloride, polyethyleneimine, and N,N-dimethylvinylbenzylamine.
[0012] Preferably, the cross-linking agent is at least one of divinylbenzene, N,N-methylenebisacrylamide, and dimethyldiallylammonium chloride.
[0013] Preferably, the initiator is at least one of azobisisobutyronitrile, dimethyl azobisisobutyrate and potassium persulfate.
[0014] Preferably, the reaction system is a reflux reaction.
[0015] Preferably, the reaction system comprises at least one of anhydrous ethanol, acetonitrile, isopropanol and dimethyl sulfoxide.
[0016] Preferably, the method comprises the following steps:
[0017] S1: contacting the prepared anion exchange magnetic beads with Pseudospermum heterophylla cyclic peptide;
[0018] S2: After the anion exchange magnetic beads are fully in contact with Pseudospermum officinale cyclic peptide, the anion exchange magnetic beads are separated from the solution;
[0019] S3: Use an eluent to desorb the Pseudoradix heterophylla polypeptide from the anion exchange magnetic beads.
[0020] Preferably, the eluent comprises 0.1%-1% formic acid and / or 0.1%-1% trifluoroacetic acid.
[0021] Compared with the prior art, the beneficial effect of the present invention is that: the present invention provides an anion exchange magnetic bead for extracting Pseudostellaria baicalensis cyclic peptide, and the anion exchange magnetic bead is prepared according to the following method: comprising the following steps:
[0022] Step 1: adding olefin-based magnetic beads, monomers, cross-linking agents and initiators to the reaction system, stirring and heating; Step 2: rapidly heating the reaction system to 70-80°C and refluxing at a constant temperature; Step 3: washing with a solvent after the reaction is completed; The present invention also relates to an application method thereof, and the anion exchange magnetic beads thus formed have selective adsorption of Pseudostellaria heterophylla cyclic peptide through modification groups on the magnetic silicon-based or polymer-based surfaces, and have high recovery rate of the adsorbed polypeptide, high efficiency, high reusability, and good application prospects.
[0023] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. The specific implementation of the present invention is given in detail by the following embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:
[0025] Figure 1 This is an electron microscope image under infrared (PS-pVIM 30-40um);
[0026] Figure 2 Schematic diagram of hydrophobic conjugate-assisted ion exchange adsorption cyclic peptide;
[0027] Figure 3 This is the total ion current diagram of Pseudostellariae cyclotide B after purification;
[0028] Figure 4 This is the mass spectrum of purified Pseudostellaria heterophylla cyclic peptide B;
[0029] Figure 5 This is the HPLC chromatogram of purified Pseudostellaria baicalensis cyclic peptide B. DETAILED DESCRIPTION
[0030] The present invention is further described below in conjunction with specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.
[0031] Due to the limitations of current Pseudostellariae separation technology, the preparation of suitable magnetic beads based on the structure of Pseudostellariae cyclic peptides is the key to achieving efficient separation and enrichment, as well as the difficulty. Generally speaking, the process of preparing magnetic materials needs to pay attention to multiple aspects such as magnetic size, type of surfactant groups and group density. Pseudostellariae cyclic peptides have a molecular weight below 1000, and their structure contains a hydrophilic cyclic amide skeleton and hydrophobic residues, as well as conjugated aromatic rings, and have low solubility in water. According to its structural characteristics, the functional design of magnetic nanomaterials can use an amphiphilic conjugated monomer cross-linking copolymerization strategy to achieve highly selective separation of Pseudostellariae cyclic peptides. The anion exchange magnetic beads are prepared as follows: Step 1: adding olefin-based magnetic beads, monomers, crosslinking agents and initiators to a reaction system, stirring and heating; wherein the volume percentage of the olefin-based magnetic beads in the reaction system is 0.1-0.5%, the volume percentage of the monomers in the reaction system is 0.5-5%, the volume percentage of the crosslinking agent in the reaction system is 0.5-5%, and the volume percentage of the initiator in the reaction system is 0.1-1%; in one embodiment, the olefin-based magnetic beads, monomers, crosslinking agents and initiators are added to the reaction system, ultrasonically dispersed and transferred into a flask, and mechanically stirred for 10 minutes before heating;
[0032] Step 2: Rapidly heat the reaction system to 70-80° C. and reflux at a constant temperature; in one embodiment, the temperature may be kept constant for several hours, such as two hours;
[0033] Step 3: After the reaction is completed, wash with a solvent. It should be understood that the solvent in step 3 can be ethanol or other elution solvents commonly used in the art. Figure 1-5As shown, the present invention uses olefin-based magnetic beads as a matrix, and coats an anion exchange polymer coating through free radical polymerization. The obtained magnetic beads have a high-efficiency separation function for cyclic peptides with a molecular weight of 1000 or less, eliminating the interference of complex organic small molecules in the matrix, can be combined with preparative chromatography to further purify the polypeptide, and can be combined with a variety of detectors to achieve the identification of Pseudo-P ...
[0034] In some embodiments, the olefin-based magnetic beads are at least one of silicon-based olefin-based magnetic beads, polymer-based olefin magnetic microspheres, and wrinkled porous polymer-based olefin magnetic microspheres.
[0035] Silicon-based olefin magnetic beads (Yin Ruicheng's own production and current sales of magnetic beads, item number E22):
[0036] Polymer olefin magnetic microspheres (Yin Ruicheng's own magnetic beads, item number MPS22):
[0037] Wrinkled porous polymer olefin magnetic microspheres (Yin Ruicheng self-produced magnetic beads, FPM30 / CPM)
[0038] It should be understood that the preparation method of the olefin-based magnetic beads, which are silicon-based olefin-based magnetic beads, polymer-based olefin magnetic microspheres, and wrinkled porous polymer-based olefin magnetic microspheres, may also be other common preparation methods in the art.
[0039] In some embodiments, the monomer is at least one of 4-aminostyrene, 4-dimethylaminostyrene, N-vinylpyrrolidone, N,N-dimethylallylamine, benzylvinyltrimethylammonium chloride, allyltrimethylammonium chloride, (3-acrylamidopropyl)trimethylammonium chloride, dimethylaminopropylacrylamide, 1-vinylimidazole, 1-allylimidazole, 1-allyl-3-methylimidazole chloride, N,N,N-trimethyl-(1-(4-vinylphenyl))methylammonium chloride, polyethyleneimine, and N,N-dimethylvinylbenzylamine.
[0040] In some embodiments, the crosslinking agent is at least one of divinylbenzene, N,N-methylenebisacrylamide, and dimethyldiallylammonium chloride.
[0041] In some embodiments, the initiator is at least one of azobisisobutyronitrile, dimethyl azobisisobutyrate, and potassium persulfate.
[0042] In some embodiments, the reaction system is a reflux reaction. In some embodiments, the reaction system comprises at least one of anhydrous ethanol, acetonitrile, isopropanol, and dimethyl sulfoxide.
[0043] An application method of anion exchange magnetic beads for extracting Pseudostellaria heterophylla cyclic peptides comprises the following steps:
[0044] S1: contacting the prepared anion exchange magnetic beads with Pseudospermum heterophylla cyclic peptide;
[0045] S2: After the anion exchange magnetic beads are fully in contact with Pseudospermum officinale cyclic peptide, the anion exchange magnetic beads are separated from the solution;
[0046] S3: Use an eluent to desorb the Pseudoradix heterophylla polypeptide from the anion exchange magnetic beads.
[0047] In some embodiments, the eluent comprises 0.1%-1% formic acid and / or 0.1%-1% trifluoroacetic acid.
[0048] Example 1: Modification of 500-800nm silicon-based olefin-based magnetic beads (Si-pVIM)
[0049] 1. Add 1g of 500-800nm silicon-based olefin-based magnetic beads to 200mL of anhydrous ethanol and 800mL of acetonitrile, stir and ultrasonically disperse for 10min, add 1g of monomer 4-aminostyrene, 1g of 1-vinylimidazole, 3mL of N,N-dimethylallylamine, 5mL of cross-linking agent divinylbenzene, and 1g of initiator azobisisobutyronitrile, then continue ultrasonication until uniform dispersion, transfer to a flask, stir mechanically for 10min, and then start to heat up.
[0050] 2. Rapidly heat up to an internal temperature of 70°C and reflux at a constant temperature for 2 hours.
[0051] 3. After the reaction is completed, magnetic separation is performed, and the beads are washed 4 times with ethanol and dried to obtain anion exchange magnetic beads.
[0052] Example 2: Modification of 5um polymer-based olefin magnetic microspheres (PS-pAIM)
[0053] 1. Add 6g of 5um polymer matrix olefin magnetic microspheres to 500mL of anhydrous ethanol and 1000mL of acetonitrile, stir and ultrasonically disperse for 10min, add 5mL of monomer 4-dimethylaminostyrene, 5g of benzylvinyltrimethylammonium chloride, 10mL of 1-allylimidazole, 5mL of crosslinking agent divinylbenzene, 20g of N-methylenebisacrylamide, and 6g of initiator dimethyl azobisisobutyrate, then continue ultrasonication until uniform dispersion, transfer to a flask and mechanically stir for 10min before starting to heat up.
[0054] 2. Rapidly heat up to the internal temperature of 75°C and reflux at constant temperature for 2 hours.
[0055] 3. After the reaction is completed, magnetic separation is performed, and the beads are washed with ethanol 5 times and dried to obtain anion exchange magnetic beads.
[0056] Example 3: Modification of 5um polymer-based olefin magnetic microspheres (PS-pAMIMC)
[0057] 1. Add 6g of 5um polymer matrix olefin magnetic microspheres to 500mL of anhydrous ethanol and 1000mL of acetonitrile, stir and ultrasonically disperse for 10min, add monomer 4-aminostyrene 10g, allyltrimethylammonium chloride 10g, dimethylaminopropyl acrylamide 25g, 1-allyl-3-methylimidazole chloride 30g, cross-linking agent divinylbenzene 50mL, dimethyldiallylammonium chloride 25mL, initiator potassium persulfate 15g, continue ultrasonication until uniform dispersion, transfer to a flask and mechanically stir for 10min before starting to heat up.
[0058] 2. Rapidly heat up to the internal temperature of 80°C and reflux at a constant temperature for 2 hours.
[0059] 3. After the reaction is completed, magnetic separation is performed, and the beads are washed 4 times with ethanol and dried to obtain anion exchange magnetic beads.
[0060] Example 4: Modification of 30-40um Corrugated Porous Polymer Matrix Olefin Magnetic Microspheres (PS-pVIM)
[0061] 1. Add 8g 30-40um wrinkled polymer matrix olefin magnetic microspheres to 500mL isopropanol and 1000ml dimethyl sulfoxide, stir and ultrasonically disperse for 10min, add monomer 4-aminostyrene 10g, 1-vinylimidazole 10g, N,N-dimethylallylamine 30mL, crosslinking agent divinylbenzene 50mL, initiator azobisisobutyronitrile 10g, continue ultrasonication until uniform dispersion, transfer to a flask, stir mechanically for 10min, and then start to heat up.
[0062] 2. Rapidly heat up to the internal temperature of 75°C and reflux at constant temperature for 2 hours.
[0063] 3. After the reaction is completed, magnetic separation is performed, and the beads are washed with ethanol 5 times and dried to obtain anion exchange magnetic beads.
[0064] Example 5: Extraction of Pseudostellariae Cyclopeptide B using 500-800nm Silicon-based Polyvinyl Imidazole Magnetic Beads (Si-pVIM)
[0065] 1. Sample preparation: 20g of Pseudostellaria heterophylla was extracted to obtain an extract, 10mg of the extract was taken and dispersed with deionized water;
[0066] Preparation of Pseudostellariae Radix Extract: Weigh 20 g of Pseudostellariae Radix, grind into powder, extract with 80% ethanol by ultrasonic for 30 min, centrifuge, collect the supernatant, and obtain the extract by rotary evaporation.
[0067] 2. Magnetic solid phase extraction: add 20 mg of 500-800 nm Si-pVIM magnetic beads to the prepared sample solution, rotate and mix for 20 min, enrich Pseudomonas aeruginosa cyclotide B, separate the magnetic beads from the solution with a magnet, and discard the original solution. Wash the magnetic beads with clean water twice, then add 1 mL of 0.5% trifluoroacetic acid in acetonitrile solution, elute in an ultrasonic extractor for 15 min, and filter through a 0.22 μm filter head;
[0068] 3. Use liquid chromatography-tandem mass spectrometry to detect and calculate the extraction rate of Pseudostellaria heterophylla cyclic peptide B.
[0069] Three parallel extractions were performed, and the results are shown in Table 1. Three parallel determinations were performed every other day, and the inter-day precision and intra-day precision were calculated. The specific data are shown in Table 1.
[0070] Example 6: Extraction of Pseudostellariae Cyclopeptide B using 5um polymer matrix polyallylimidazole magnetic beads (PS-pAIM)
[0071] 1. Sample preparation: 20g of Pseudostellaria heterophylla was extracted to obtain an extract, 10mg of the extract was taken and dispersed with deionized water;
[0072] Preparation of Pseudostellariae Radix Extract: Weigh 20 g of Pseudostellariae Radix, grind into powder, extract with 80% ethanol by ultrasonic for 30 min, centrifuge, collect the supernatant, and obtain the extract by rotary evaporation.
[0073] 2. Magnetic solid phase extraction: add 20 mg of 5um polymer matrix PS-pAIM magnetic beads to the prepared sample solution, rotate and mix for 20 minutes, enrich Pseudomonas aeruginosa cyclotide B, separate the magnetic beads from the solution with a magnet, and discard the original solution. Wash the magnetic beads with clean water twice, then add 1 mL of 0.5% trifluoroacetic acid in acetonitrile solution, elute in an ultrasonic extractor for 15 minutes, and filter through a 0.22μm filter head;
[0074] 3. Use liquid chromatography-tandem mass spectrometry to detect and calculate the extraction rate of Pseudostellaria heterophylla cyclic peptide B.
[0075] Three parallel extractions were performed, and the results are shown in Table 1. Three parallel determinations were performed every other day, and the inter-day precision and intra-day precision were calculated. The specific data are shown in Table 1.
[0076] Example 7: Extraction of Pseudostellariae Cyclopeptide B Using 5um Polymer Matrix Poly(1-allyl-3-methylimidazole) Chloride Magnetic Beads (PS-pAMIMC)
[0077] 1. Sample preparation: 20g of Pseudostellaria heterophylla was extracted to obtain an extract, 10mg of the extract was taken and dispersed with deionized water;
[0078] Preparation of Pseudostellariae Radix Extract: Weigh 20 g of Pseudostellariae Radix, grind into powder, extract with 80% ethanol by ultrasonic for 30 min, centrifuge, collect the supernatant, and obtain the extract by rotary evaporation.
[0079] 2. Magnetic solid phase extraction: add 20 mg of 5um polymer matrix PS-pAMIMC magnetic beads to the prepared sample solution, rotate and mix for 20 minutes, enrich Pseudo-P ...
[0080] 3. Use liquid chromatography-tandem mass spectrometry to detect and calculate the extraction rate of Pseudostellaria heterophylla cyclic peptide B.
[0081] Three parallel extractions were performed, and the results are shown in Table 1. Three parallel determinations were performed every other day, and the inter-day precision and intra-day precision were calculated. The specific data are shown in Table 1.
[0082] Example 8: Extraction of Pseudostellariae Cyclopeptide B Using 30-40um Corrugated Porous Polymer Matrix Polyvinyl Imidazole Magnetic Beads (PS-pVIM)
[0083] 1. Sample preparation: 20g of Pseudostellaria heterophylla was extracted to obtain an extract, 10mg of the extract was taken and dispersed with deionized water;
[0084] Preparation of Pseudostellariae Radix Extract: Weigh 20 g of Pseudostellariae Radix, grind into powder, extract with 80% ethanol by ultrasonic for 30 min, centrifuge, collect the supernatant, and obtain the extract by rotary evaporation.
[0085] 2. Magnetic solid phase extraction: add 20 mg of 30-40 μm corrugated porous polymer matrix PS-pVIM magnetic beads to the prepared sample solution, rotate and mix for 20 minutes, enrich Pseudostigma cyclotide B, separate the magnetic beads from the solution with a magnet, and discard the original solution. Wash the magnetic beads with clean water twice, then add 1 mL of 0.5% trifluoroacetic acid in acetonitrile solution, elute in an ultrasonic extractor for 15 minutes, and filter through a 0.22 μm filter head;
[0086] 3. Use liquid chromatography-tandem mass spectrometry to detect and calculate the extraction rate of Pseudostellaria heterophylla cyclic peptide B.
[0087] Three parallel extractions were performed, and the results are shown in Table 1. Three parallel determinations were performed every other day, and the inter-day precision and intra-day precision were calculated. The specific data are shown in Table 1.
[0088] Comparative Example 1
[0089] Commercially available magnetic beads: Manufacturer: Suzhou Yingruicheng Biochemical Technology Co., Ltd.; Model: MSi050 / PLS silica-based lipophilic PLS purification magnetic beads.
[0090] Table 1 Extraction rate and precision of Pseudostellariae Cyclopeptide B from Pseudostellariae Radix (n=3)
[0091]
[0092]
[0093] It can be seen from Table 1 that the anion exchange magnetic beads of the present invention can efficiently and highly selectively separate the cyclic peptides in Pseudostellariae Radix, and both the inter-day RSD and the intra-day RSD are less than 5%, indicating that the method of the present invention has high precision.
[0094] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the embodiments shown here.
Claims
1. An anion exchange magnetic bead for extracting Pseudostellaria heterophylla cyclic peptide, characterized in that: The anion exchange magnetic beads are prepared as follows: Step 1: adding olefin-based magnetic beads, monomers, cross-linking agents and initiators to the reaction system, stirring and heating; wherein the volume percentage of the olefin-based magnetic beads in the reaction system is 0.1-0.5%, the volume percentage of the monomers in the reaction system is 0.5-5%, the volume percentage of the cross-linking agent in the reaction system is 0.5-5%, and the volume percentage of the initiator in the reaction system is 0.1-1%; Step 2: Rapidly heat the reaction system to 70-80°C and reflux at a constant temperature; Step 3: After the reaction is completed, wash with a solvent.
2. The anion exchange magnetic beads for extracting Pseudostellariae cyclic peptide according to claim 1, characterized in that: The olefin-based magnetic beads are at least one of silicon-based olefin magnetic beads, polymer olefin magnetic microspheres, and wrinkled porous polymer olefin magnetic microspheres.
3. The anion exchange magnetic beads for extracting Pseudostellariae cyclic peptide according to claim 1, characterized in that: The monomer is at least one of 4-aminostyrene, 4-dimethylaminostyrene, N-vinyl pyrrolidone, N,N-dimethylallylamine, benzylvinyltrimethylammonium chloride, allyltrimethylammonium chloride, (3-acrylamidopropyl)trimethylammonium chloride, dimethylaminopropylacrylamide, 1-vinylimidazole, 1-allylimidazole, 1-allyl-3-methylimidazole chloride, N,N,N-trimethyl-(1-(4-vinylphenyl))methylammonium chloride, polyethyleneimine, and N,N-dimethylvinylbenzylamine.
4. The anion exchange magnetic beads for extracting Pseudostellariae cyclic peptide according to claim 1, characterized in that: The crosslinking agent is at least one of divinylbenzene, N,N-methylenebisacrylamide and dimethyldiallylammonium chloride.
5. The anion exchange magnetic beads for extracting Pseudostellariae cyclic peptide according to claim 1, characterized in that: The initiator is at least one of azobisisobutyronitrile, dimethyl azobisisobutyrate and potassium persulfate.
6. The anion exchange magnetic beads for extracting Pseudostellariae cyclic peptide according to claim 1, characterized in that: The reaction system is a reflux reaction.
7. The anion exchange magnetic beads for extracting Pseudostellariae cyclic peptide according to claim 6, characterized in that: The reaction system comprises at least one of anhydrous ethanol, acetonitrile, isopropanol and dimethyl sulfoxide.
8. A method for using anion exchange magnetic beads for extracting Pseudostellariae cyclic peptides as claimed in claim 1, characterized in that: The following steps are involved: S1: contacting the prepared anion exchange magnetic beads with Pseudospermum heterophylla cyclic peptide; S2: After the anion exchange magnetic beads are fully in contact with Pseudospermum officinale cyclic peptide, the anion exchange magnetic beads are separated from the solution; S3: Use an eluent to desorb the Pseudoradix heterophylla polypeptide from the anion exchange magnetic beads.
9. The application method of anion exchange magnetic beads for extracting Pseudostellariae cyclic peptides as claimed in claim 8, characterized in that: The eluent includes 0.1%-1% formic acid and / or 0.1%-1% trifluoroacetic acid.