High-purity monkshood polysaccharide and preparation method thereof

Abstract

The invention provides high-purity monkshood polysaccharide and a preparation method thereof. The preparation method comprises the following steps: carrying out drying, crushing, ultrasonic stirring, heating stirring, boiling, centrifugation, concentration, alcohol precipitation, resuspension, centrifugation, suction filtration and vacuum drying on rhizoma typhonii slices to obtain a radix aconiti carmichaeli polysaccharide crude product; completely dissolving the radix aconiti carmichaeli polysaccharide crude product with water, and separating polysaccharide by using a membrane filtration system to obtain a radix aconiti carmichaeli polysaccharide solution; and finally, removing impurities in the monkshood polysaccharide solution through an affinity purification column of the monkshood polysaccharide nucleic acid aptamer, and freeze-drying to obtain the high-purity monkshood polysaccharide. The dissolution rate of radix aconiti carmichaeli polysaccharide is improved through ultrasonic stirring and heating stirring; performing boiling, concentration, alcohol precipitation, resuspension and freeze drying to obtain a high-purity radix aconiti carmichaeli polysaccharide crude product; then screening specific monkshood polysaccharide by adopting a membrane filtration system; finally, alkaloid is accurately and efficiently removed by using an aptamer, and the purity of the monkshood polysaccharide is improved.

Description

Technical Field

[0001] The present invention relates to the field of detection of aconite polysaccharides, and specifically relates to a high-purity aconite polysaccharide and a preparation method thereof. Background Art

[0002] Aconite was first recorded in "Shennong Ben Cao Jing" and is a processed product of the lateral roots of the perennial herbaceous plant Aconitum carmichaeli Debx. of the Ranunculaceae family. It is currently widely cultivated in regions such as Sichuan, Shaanxi, and Guizhou in China. It is a traditional Chinese medicine with a very long history of use in China, and has the effects of restoring yang and rescuing from collapse, tonifying fire and assisting yang, dispelling cold and relieving pain, and can be used for symptoms of collapse of yang and deficiency, with faint pulse and cold limbs. Its main chemical components include alkaloids, flavonoids, polysaccharides, saponins, sterols, fatty acids, inorganic salts, etc. Before the mid-20th century, the research on the material basis of aconite mainly focused on alkaloids. It was not until recent years that the research on aconite polysaccharides has gradually attracted people's attention.

[0003] In recent years, relevant research has found that aconite polysaccharide, as an active substance in aconite, has very extensive pharmacological activities, such as immunomodulation, anti-tumor, anti-depression, organ protection, blood glucose regulation, and anti-inflammatory and antibacterial effects. And it has fewer adverse reactions and higher safety. However, due to the complex components of traditional Chinese medicine aconite, it is often difficult to improve the purity of its polysaccharides, and the existing alkaloids (mainly aconitine, hypaconitine, and mesaconitine) will greatly limit the application of aconite polysaccharides.

[0004] Currently, the preparation methods of aconite polysaccharides mainly include enzyme-assisted extraction, ultrasonic extraction, ultrasonic-assisted thermal extraction, water immersion extraction, microwave extraction, and water extraction and alcohol precipitation extraction. However, the above methods have limitations in the preparation of aconite polysaccharides, such as large differences in extraction methods, low extraction efficiency, alkaloid residues, and difficult polysaccharide purification, resulting in large losses of polysaccharides. This causes the polysaccharide yield obtained by the above methods to be less than 50%. Therefore, there is an urgent need for a method to improve the purity of aconite polysaccharides.

[0005] Nucleic acid aptamers have a wide range of applications, especially in the biomedical field, such as as diagnostic tools, targeted drugs in clinical treatment, food and environmental monitoring, and vaccine development. Nucleic acid aptamers are single-stranded nucleic acid molecules (DNA or RNA) selected from a large number of random sequences through a screening process, and they can bind specifically to specific target molecules (such as proteins, small molecules, or cells). By constructing a single-stranded random oligonucleotide library and using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology for multiple enrichments and screenings, nucleic acid aptamers with high specificity and affinity for the target can be preferentially selected in vitro, thus avoiding the difficulties brought by in vivo immune reactions. The working principle of the aptamer affinity column is to utilize the selective adsorption of the aptamer to the target molecule to achieve the extraction and purification of the target molecule in complex samples, and this adsorption is reversible. The purification of the aptamer affinity column has become an important development direction. Summary of the Invention

[0006] In view of the problem that the polysaccharide yield extracted by the existing technology is less than 50%, the present invention provides a high-purity aconite polysaccharide and a preparation method thereof.

[0007] The technical method of the present invention is as follows:

[0008] A preparation method of high-purity aconite polysaccharide, comprising:

[0009] S1. Dry and crush the processed aconite slices to obtain aconite granules;

[0010] S2. Perform ultrasonic stirring on the aconite granules and water, and filter to obtain the first filtrate;

[0011] S3. Heat and stir the aconite granules and water after step S2, and filter to obtain the second filtrate;

[0012] S4. Boil, centrifuge, and concentrate the first filtrate and the second filtrate to obtain a concentrated solution;

[0013] S5. Add ethanol to the concentrated solution, and obtain an ethanol precipitation precipitate after ethanol precipitation;

[0014] S6. Use absolute ethanol to resuspend and centrifuge the ethanol precipitation precipitate for multiple times to obtain a resuspended precipitate, and the number of times is greater than or equal to 2;

[0015] S7. Filter the resuspended precipitate by suction filtration and vacuum dry it to obtain the crude aconite polysaccharide;

[0016] S8. Completely dissolve the crude aconite polysaccharide in water, and use a membrane filtration system to separate the polysaccharide to obtain an aconite polysaccharide solution;

[0017] S9. Remove impurities in the polysaccharide solution of Aconitum carmichaelii Debx. through an affinity purification column of Aconitum carmichaelii Debx. polysaccharide nucleic acid aptamer, and then obtain high-purity polysaccharide of Aconitum carmichaelii Debx. through freeze-drying.

[0018] In the step S1, the particle size of the Aconitum carmichaelii Debx. particles is 15 - 20 mesh.

[0019] In the step S2, the solid-liquid ratio of the Aconitum carmichaelii Debx. particles to water is 1:8 - 12, the power of ultrasonic wave is 400 - 600 W, and the time of ultrasonic wave is 8 - 12 hours.

[0020] In the step S3, the heating temperature is 70 - 80 °C, the stirring time is 4 - 6 hours, and the solid-liquid ratio of the Aconitum carmichaelii Debx. particles to water after the step S2 is 1:8 - 12.

[0021] In the step S4, the boiling time is greater than or equal to 1 hour, the boiling temperature is 90 - 110 °C; the centrifugation speed is 15000 - 20000 rpm / min, the centrifugation time is 10 - 20 minutes, and the volume ratio before and after concentration is 11 - 14:1.

[0022] In the step S5, the alcohol content of ethanol is greater than or equal to 80%, the time of alcohol precipitation is 8 - 10 hours, and the temperature of alcohol precipitation is 2 - 4 °C.

[0023] In the step S6, the centrifugation speed is 3500 - 4000 rpm / min, and the centrifugation time is 10 - 20 minutes.

[0024] In the step S8, the filler of the affinity purification column of Aconitum carmichaelii Debx. polysaccharide nucleic acid aptamer is agarose modified with polyethylene glycol as the carrier, and then the nucleic acid aptamer with the nucleic acid sequence such as SEQ ID NO.1 is covalently coupled with the carrier to obtain. Specifically, the preparation steps of the Aconitum carmichaelii Debx. polysaccharide aptamer affinity purification column include:

[0025] A1. Prepare agarose modified with polyethylene glycol, and then swell and wash with agarose modified with polyethylene glycol as the carrier;

[0026] A2. Prepare an amino-modified aptamer, and then renature the amino-modified aptamer;

[0027] A3. Couple, block, wash, and pack the agarose modified with polyethylene glycol processed in the step A1 with the amino-modified aptamer processed in the step A2 to obtain the Aconitum carmichaelii Debx. polysaccharide aptamer affinity purification column.

[0028] In the step S9, the membrane filtration system separates and purifies polysaccharides with a molecular weight of 3000 - 100000 Da.

[0029] The present invention also provides a high-purity polysaccharide of Aconitum carmichaelii Debx., and the high-purity polysaccharide of Aconitum carmichaelii Debx. is prepared by the above preparation method.

[0030] The beneficial effects of the present invention are as follows:

[0031] 1. The present invention improves the dissolution rate of aconite polysaccharide by ultrasonic stirring and heating stirring; then, through boiling, concentration, ethanol precipitation, resuspension, and freeze-drying, high-purity crude aconite polysaccharide is obtained, and the temperature is controlled during this process to prevent the polysaccharide structure from being damaged by high temperature; subsequently, ultrafiltration is used to screen aconite polysaccharide with a specific molecular weight and biological activity; finally, aptamers are used to accurately and efficiently remove alkaloids, improving the purity of aconite polysaccharide. The present invention provides a broad application prospect for the development and utilization of aconite polysaccharide.

[0032] 2. The nucleic acid aptamer involves the coordination among chemistry, biochemistry, molecular biology and other fields in the screening application of polysaccharide molecules, aiming to better prepare high-purity aconite polysaccharide. The present invention breakthroughly uses nucleic acid aptamers, and adopts the method of capturing polysaccharides with a nucleic acid aptamer affinity purification column and eluting impurities, effectively solving the problems of alkaloid residue, low efficiency, and long time consumption in aconite polysaccharide extracts.

[0033] 3. The present invention uses aconite polysaccharide aptamer and polyethylene glycol-modified agarose to prepare an aconite polysaccharide aptamer affinity purification column, which is simple to prepare, low in price, and can be reused multiple times, with the number of uses exceeding 25 times.

[0034] 4. The present invention has high efficiency in purifying aconite polysaccharide, and the recovery rate of aconite polysaccharide is above 99%. Brief Description of the Drawings

[0035] Figure 1 It is a schematic structural diagram of an aconite polysaccharide aptamer affinity purification column;

[0036] Figure 2 It is a schematic diagram of purifying polysaccharide using an affinity purification column coupled with polysaccharide nucleic acid aptamer;

[0037] Figure 3 It is a standard glucose curve diagram made by the phenol-sulfuric acid method in the present invention;

[0038] Figure 4 is the chromatogram result of detecting the content of total aconitine (aconitine, hypaconitine, mesaconitine) in the polysaccharide product by high performance liquid chromatography in the present invention; among them, Figure 4-1 It is the result diagram of detecting the total aconitine of crude aconite polysaccharide, Figure 4-2 It is the result diagram of detecting the total aconitine of high-purity aconite polysaccharide purified without using nucleic acid aptamer, Figure 4-3 It is the total aconitine mixed standard product diagram of high-purity aconite polysaccharide purified using nucleic acid aptamer (source: National Institutes for Food and Drug Control, batch number 112029-202302).

[0039] Reference Signs:

[0040] 1 - Injection port plug, 2 - Cylinder, 3 - Upper sieve plate, 4 - Carrier packing, 5 - Lower sieve plate, 6 - Sampling tube. Detailed implementation mode

[0041] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the scope of protection of the present invention.

[0042] The present invention provides a method for preparing high-purity aconite polysaccharide, including:

[0043] S1. Dry and crush the processed aconite slices to obtain aconite granules.

[0044] In one embodiment, the processed aconite slices are a kind of aconite, mainly produced in provinces such as Sichuan and Shaanxi.

[0045] In step S1, the crushing is carried out using a conventional crushing machine in the art to crush the processed aconite slices. The particle size of the aconite granules is 15 - 20 mesh.

[0046] S2. Perform ultrasonic stirring on the aconite granules and water, and filter to obtain the first filtrate.

[0047] In step S2, the water is pure water or sterile injection water.

[0048] In step S2, the solid-liquid ratio of the aconite granules and water is 1:8 - 12. For example, the solid-liquid ratio can be 1:10, 1:11.

[0049] In step S2, the power of the ultrasonic wave is 400 - 600W. For example, the power of the ultrasonic wave can be 450W, 500W, 550W. The time of the ultrasonic wave is 8 - 12 hours. For example, the time of the ultrasonic wave can be 11 hours, 12 hours.

[0050] S3. Heat and stir the aconite granules and water after step S2, and filter to obtain the second filtrate.

[0051] In step S3, the heating temperature is 70 - 80°C. For example, the heating temperature is 72°C, 77°C, 79°C. The stirring time is 4 - 6 hours. For example, the stirring time can be 4, 5, 6 hours.

[0052] In step S3, the solid-liquid ratio of the aconite granules and water after step S2 is 1:8 - 12. For example, the solid-liquid ratio of the aconite granules and water after step S2 is 1:13.

[0053] S4. Boil, centrifuge, and concentrate the first filtrate and the second filtrate to obtain a concentrated solution.

[0054] In step S4, the boiling time is greater than or equal to 1 hour. The boiling temperature is 90 - 110 °C. Preferably, the boiling time can be 2 h or 3 h. The boiling temperature can be 100 °C.

[0055] In step S4, the centrifugation speed is 15000 - 20000 rpm / minute, and the centrifugation time is 10 - 20 minutes. For example, the centrifugation speed can be 16000 rpm / minute, 18000 rpm / minute, 19000 rpm / minute, and the centrifugation time can be 12 minutes, 15 minutes, 17 minutes. Centrifugation is carried out using a tubular centrifuge at room temperature.

[0056] In step S4, the volume ratio before and after concentration is 11 - 14:1. For example, the volume ratio before and after concentration is 13:1.

[0057] S5. Add ethanol to the concentrated solution, and obtain an alcohol-precipitated precipitate after alcohol precipitation.

[0058] In step S5, the alcohol content of ethanol is greater than or equal to 80%, preferably 95%. Here, the volume of 95% ethanol added to the concentrated solution is 4 - 6 times that of the concentrated solution.

[0059] In step S5, the alcohol precipitation time is 8 - 10 hours, and the alcohol precipitation temperature is 2 - 4 °C. Preferably, the alcohol precipitation time is 9 hours, and the alcohol precipitation temperature is 3 °C.

[0060] S6. Resuspend and centrifuge the alcohol-precipitated precipitate with anhydrous ethanol multiple times to obtain a resuspended precipitate, and the number of times is greater than or equal to 2.

[0061] In step S6, resuspension can elute impurities and make the polysaccharide precipitation more uniform. The ratio of the alcohol-precipitated precipitate to anhydrous ethanol is 1:2 - 4. Preferably, the ratio of the alcohol-precipitated precipitate to anhydrous ethanol is 1:3.

[0062] In step S6, the centrifugation speed is 3500 - 4000 rpm / minute, and the centrifugation time is 10 - 20 minutes.

[0063] S7. Filter the resuspended precipitate by suction filtration and vacuum dry it to obtain the crude aconite polysaccharide.

[0064] In step S7, suction filtration is carried out using conventional suction filtration equipment in the art.

[0065] In step S7, the vacuum drying time is 5 - 8 hours; the vacuum drying temperature is -30 to -40 °C.

[0066] S8. Completely dissolve the crude polysaccharide of Aconitum carmichaelii Debx. in water, and use a membrane filtration system to separate the polysaccharide to obtain an Aconitum carmichaelii Debx. polysaccharide solution.

[0067] In step S8, the water is pure water or sterile injection water.

[0068] In step S8, the membrane filtration system separates and purifies polysaccharides with a molecular weight of 3000 - 100000 Da. The membrane filtration system adopts a conventional membrane filtration system in the art.

[0069] S9. Pass the Aconitum carmichaelii Debx. polysaccharide solution through an affinity purification column of the aptamer of Aconitum carmichaelii Debx. polysaccharide to remove impurities, and then freeze-dry to obtain high-purity Aconitum carmichaelii Debx. polysaccharide.

[0070] In step S9, the impurities include aconitine, protein, cellulose, etc. The filler of the affinity purification column of the aptamer of Aconitum carmichaelii Debx. polysaccharide is obtained by covalently coupling an aptamer with a nucleic acid sequence such as SEQ ID NO.1 to agarose modified with polyethylene glycol as a carrier. Here, the agarose modified with polyethylene glycol in the present invention undergoes covalent coupling with the amino-modified aptamer, and the coupling product is stable and has a high coupling rate. The used aptamer of nucleic acid is obtained by an in vitro chemical synthesis method, which can ensure the correctness of the sequence and the consistency between batches, and greatly reduces the differences between different batches.

[0071] The present invention makes full use of the advantages of high specificity and high affinity of the aptamer of nucleic acid. By using the aptamer of Aconitum carmichaelii Debx. polysaccharide to specifically bind the Aconitum carmichaelii Debx. polysaccharide in the sample, the purification efficiency of the affinity purification column is greatly improved. The aptamer of nucleic acid in the present invention is less affected by the operating environment and organic solvents, and is especially suitable for purification at the production line sites of various industries.

[0072] Specifically, the preparation steps of the affinity purification column of the aptamer of Aconitum carmichaelii Debx. polysaccharide include: A1. Prepare agarose modified with polyethylene glycol, and then swell and wash it with agarose modified with polyethylene glycol as a carrier; A2. Prepare an amino-modified aptamer, and then renature the amino-modified aptamer; A3. Couple, block, wash, and pack the agarose modified with polyethylene glycol treated in step A1 with the amino-modified aptamer treated in step A2 to obtain an affinity purification column of the aptamer of Aconitum carmichaelii Debx. polysaccharide.

[0073] As Figure 1 shown, the structure of the affinity purification column of the aptamer of Aconitum carmichaelii Debx. polysaccharide includes a sample inlet plug 1, a column body 2, an upper sieve plate 3 (upper sieve plate), a carrier filler 4, a lower sieve plate 5 (lower sieve plate), and a sample receiving tube 6.

[0074] Specifically, step A1 includes: A101 preparing polyethylene glycol-modified agarose: Mix agarose with an equal volume of water, add it to a reactor equipped with a pH electrode and a magnetic stirrer, add polyethylene glycol to the above agarose solution in an amount of 50-300 mg of polyethylene glycol per mL of agarose solution, adjust the pH value to 11 ± 0.1 with NaOH, control the whole reaction pH value at 11 ± 0.1, control the temperature at 20°C ± 5°C, and complete the reaction in 3-12 minutes; after the reaction, quickly add an equal volume of ice chips to the above reaction solution, and quickly pour it into a Buchner funnel, and filter and wash it with a cold buffer solution 10-15 times the volume of the agarose solution. The hydroxyl groups on its surface react with polyethylene glycol to obtain polyethylene glycol-modified agarose; A102 swelling and washing of the carrier: Using polyethylene glycol-modified agarose as the carrier, soak 30-150 mg of the carrier powder in 1-5 mL of 1 mM hydrochloric acid for 0.5-1 hour for swelling; wash the swollen carrier with 1 mM hydrochloric acid 3-6 times, with 2-5 mL of hydrochloric acid used each time, then wash it with distilled water 2-5 times, with 1-5 mL of distilled water used each time, and finally wash it with Na2HPO4 buffer solution 2-5 times, with 1-5 mL of buffer solution used each time.

[0075] Specifically, step A2 includes: A201 preparing amino-modified aptamer: Covalently connect a C7 spacer arm -(CH2)7- or a C6 spacer arm -(CH2)6- to the 3' or 5' end of the nucleic acid aptamer, and then covalently modify an amino group at the end of the C7 spacer arm or the C6 spacer arm to obtain an amino-modified aptamer; A202 renaturation of the amino-modified aptamer: Dissolve 1-50 D of the amino-modified aptamer in 200-1000 μL of Na2HPO4 buffer solution, renature it at 75-95°C for 3-5 minutes, and then place it at room temperature for 15-60 minutes.

[0076] Specifically, step A3 includes: A301 Coupling: Add 200 - 1000 μL of the amino - modified aptamer after treatment with A2 to the polyethylene glycol - modified agarose after treatment with A1, and shake overnight on a shaker at 30°C; A302 Blocking: Wash the coupling product obtained in A301 successively with 200 mM Na2HPO4 aqueous solution at pH 8.0 for 2 - 5 times, 15 mL each time, then wash with 0.1 M Tris - HCl buffer at pH 8.0 for 2 - 5 times, 1 - 5 mL each time, and then add 2 - 5 mL of 0.1 M Tris - HCl solution, shake and react on a shaker at 30°C for 1 - 6 h to block the remaining active sites, obtaining the carrier - aptamer coupling gel; A303 Washing: Wash the carrier - aptamer coupling gel obtained in A302 successively with acetic acid buffer and Tris - HCl buffer alternately for 3 - 5 times, and the amount of acetic acid buffer or Tris - HCl buffer used for each wash is 1 - 5 mL to remove the uncoupled aptamers; Resuspend the washed coupling gel with 1 - 5 mL of binding buffer, and the obtained coupling gel suspension is ready for column packing; wherein, the acetic acid buffer is 0.1 M acetic acid - sodium acetate aqueous solution containing 0.5 M NaCl, pH 4.0; the Tris - HCl buffer concentration is 0.1 M containing 0.5 M NaCl, pH 8.0; the binding buffer contains 10 mM Tris, 120 mM NaCl, 5 mM KCl and 5 mM MgCl2, pH 7.5; A304 Column Packing: Take a solid - phase extraction column with a volume of 1 - 5 mL, pack the column with the above - mentioned coupling gel suspension until the height of the gel is 1 cm, add 0.5 - 3 mL of 0.05 w / v% NaN3 solution, and store at 4°C.

[0077] The usage method is as follows: 1) Take out the affinity purification column of aconitum polysaccharide aptamer, open the inlet plug, connect the inlet to the syringe barrel, and connect the syringe to the pneumatic operation rack. 2) Open the outlet plug, balance the affinity purification column with 5 mL of binding buffer, adjust the air pump pressure of the air hole operation rack to make the liquid flow out at a rate of 3 drops / second. 3) Add the above - mentioned complex solution to the affinity purification column and adjust the flow rate to 1 - 2 drops / second until all the samples flow out of the affinity purification column. 4) Wash the affinity purification column with 1 mL of binding buffer. 5) Add 1 mL of purified water and collect the elution product.

[0078] As Figure 2 It is a schematic diagram of purifying polysaccharides using an affinity purification column coupled with polysaccharide nucleic acid aptamers, and aconitum polysaccharides are extracted through the affinity purification column.

[0079] In step S8, the freeze - drying time is 5 - 8 hours, and the temperature is - 30°C - - 40°C.

[0080] In the present invention, without special instructions, all preparation raw materials are commercially available products well - known to those skilled in the art.

[0081] Example 1: Crude Aconite Polysaccharide

[0082] S1. After drying the processed aconite roots, crush the dried aconite roots into granules with a pulverizer, and then pass through a 15-20 mesh sieve to obtain aconite granules.

[0083] S2. Weigh the aconite granules, and ultrasonically stir the aconite granules with pure water at room temperature for 12 hours, then take the filtrate (i.e., the extract). The solid-liquid ratio of aconite granules to pure water is 1:8-12, the power of ultrasonic wave is 400-600W, and the ultrasonic time is 8-12 hours.

[0084] S3. When the water temperature reaches a constant temperature of 70-80 °C, stir the aconite granules again for 4 hours, then take the filtrate (i.e., the extract). The solid-liquid ratio of aconite granules to pure water is 1:8-12.

[0085] S4. Filter the extract twice and then boil it for at least 1 hour. Use a tubular centrifuge to centrifuge the filtrate at a speed of 19000 rpm / min at room temperature, and concentrate the medicinal liquid under vacuum with a concentration ratio of 13:1.

[0086] S5. Add 95% ethanol with a volume 4-6 times that of the concentrated liquid to the concentrated medicinal liquid, and carry out alcohol precipitation at a constant temperature of 4 °C for 8-10 hours.

[0087] S6. Resuspend the precipitate with absolute ethanol and centrifuge at a speed of 3800 r / min for 15 minutes.

[0088] S7. Repeat step S6 twice.

[0089] S8. Filter and vacuum dry the precipitate after the treatment in step S7 to obtain the crude polysaccharide.

[0090] Example 2: Preparation of an aptamer affinity purification column for aconite polysaccharide aptamer

[0091] 1) Preparation of polyethylene glycol-modified agarose: Mix agarose with an equal volume of water, add it to a reactor equipped with a pH electrode and a magnetic stirrer, add polyethylene glycol to the above agarose solution in an amount of 50-300 mg of polyethylene glycol per mL of agarose solution, adjust the pH value to 11 ± 0.1 with NaOH, control the whole reaction pH value at 11 ± 0.1, control the temperature at 20 °C ± 5 °C, and the reaction is completed in 3-12 minutes; after the reaction, quickly add an equal volume of ice chips to the above reaction solution, and quickly pour it into a Buchner funnel, and filter and wash it with a cold buffer solution 10-15 times the volume of the agarose solution. The hydroxyl groups on its surface react with polyethylene glycol to obtain polyethylene glycol-modified agarose.

[0092] 2) Swelling and washing of the carrier: Using polyethylene glycol-modified agarose as the carrier, soak 30 - 150 mg of the carrier powder in 1 - 5 mL of 1 mM hydrochloric acid for 0.5 - 1 hour for swelling; wash the swollen carrier 3 - 6 times with 1 mM hydrochloric acid, with 2 - 5 mL of hydrochloric acid used each time, then wash it 2 - 5 times with distilled water, with 1 - 5 mL of distilled water used each time, and finally wash it 2 - 5 times with Na2HPO4 buffer solution, with 1 - 5 mL of buffer solution used each time.

[0093] 3) Preparation of amino-modified aptamer: Covalently connect the C7 spacer arm -(CH2)7- or C6 spacer arm -(CH2)6- to the 3’ or 5’ end of the nucleic acid aptamer, and then covalently modify an amino group at the end of the C7 spacer arm or C6 spacer arm to obtain the amino-modified aptamer.

[0094] 4) Aptamer renaturation: Dissolve 1 - 5 OD of the 3’ or 5’ end amino-modified aconitic polysaccharide aptamer-specific DNA in 200 - 1000 μL of Na2HPO4 buffer solution, renature it at 75 - 95 °C for 3 - 5 minutes, and then leave it at room temperature for 15 - 60 minutes.

[0095] 5) Coupling: Add 200 - 1000 μL of the aptamer solution renatured in step 4) to the carrier washed in step 2), and shake it overnight on a shaker at 30 °C.

[0096] 6) Blocking: Wash the coupling product obtained in step 5) successively 2 - 5 times with 200 mM Na2HPO4 aqueous solution at pH 8.0, with 15 mL used each time, then wash it 2 - 5 times with 0.1 M Tris-HCl buffer solution at pH 8.0, with 1 - 5 mL used each time, and then add 2 - 5 mL of 0.1 M Tris-HCl solution, shake and react it on a shaker at 30 °C for 1 - 6 h to block the remaining active sites, obtaining the carrier-aptamer conjugate gel.

[0097] 7) Washing: Wash the above carrier-aptamer conjugate gel successively 3 - 5 times alternately with acetic acid buffer solution and Tris-HCl buffer solution, with 1 - 5 mL of acetic acid buffer solution or Tris-HCl buffer solution used each time for washing to remove the uncoupled aptamer; resuspend the washed conjugate gel with 1 - 5 mL of binding buffer solution, and the obtained conjugate gel suspension is ready to be packed into a column. Among them, the acetic acid buffer solution is 0.1 M acetic acid-sodium acetate aqueous solution containing 0.5 M NaCl, pH 4.0; the Tris-HCl buffer solution concentration is 0.1 M containing 0.5 M NaCl, pH 8.0; the binding buffer solution contains 10 mM Tris, 120 mM NaCl, 5 mM KCl and 5 mM MgCl2, pH 7.5.

[0098] 8) Column packing: Take a solid-phase extraction column with a volume of 1 - 5 mL, pad the lower sieve plate, and pack the column with the above-mentioned coupled colloid suspension until the height of the colloid is 1 cm. Then add 0.5 - 3 mL of 0.05 w / v% NaN3 solution and store it at 4°C.

[0099] High-purity aconite polysaccharide without using nucleic acid aptamer purification in Example 3

[0100] S1. After drying the processed aconite slices, use a pulverizer to crush the dried aconite slices into granules, and then pass them through a 15 - 20 mesh sieve to obtain aconite granules.

[0101] S2. Weigh the aconite granules, and ultrasonically stir the aconite granules with pure water at room temperature for 12 hours, then take the filtrate (i.e., the extract). The solid-liquid ratio of aconite granules to pure water is 1:8 - 12, and the power of ultrasonic wave is 400 - 600 W.

[0102] S3. When the water temperature reaches a constant temperature of 70 - 80°C, stir the aconite granules again for 4 hours, then take the filtrate (i.e., the extract). The solid-liquid ratio of aconite granules to pure water is 1:8 - 12.

[0103] S4. After filtering the extract twice, boil it for at least 1 hour, and use a tubular centrifuge to centrifuge the filtrate at a speed of 19000 rpm / min at room temperature, and vacuum-concentrate the medicinal liquid according to a concentration ratio of 13:1.

[0104] S5. Add 95% ethanol with a volume 4 - 6 times that of the concentrated liquid to the concentrated medicinal liquid to make the alcohol content of the alcohol precipitation solution reach more than 80%, and keep it at a constant temperature of 4°C for alcohol precipitation for 8 - 10 hours.

[0105] S6. Resuspend the precipitate with absolute ethanol and centrifuge it at a speed of 3800 r / min for 15 minutes.

[0106] S7. Repeat step S6 twice.

[0107] S8. Filter the precipitate processed in step S7 by suction and vacuum-dry it to obtain the crude polysaccharide.

[0108] S9. Weigh a certain mass of the crude polysaccharide and completely dissolve it with sterilized injection water to obtain the crude aconite polysaccharide solution.

[0109] S10. Use a membrane filtration system to separate and purify the polysaccharide solution with a molecular weight of 3000 - 100000 Da after freeze-drying the ultrafiltered polysaccharide solution to obtain high-purity aconite polysaccharide.

[0110] High-purity aconite polysaccharide using nucleic acid aptamer purification in Example 4

[0111] S1. After drying the processed aconite slices, use a pulverizer to crush the dried aconite slices into granules, and then pass them through a 15 - 20 mesh sieve to obtain aconite granules;

[0112] S2. Weigh the aconite granules. After ultrasonic stirring the aconite granules with pure water at room temperature for 12 hours, take the filtrate (i.e., the extract). The solid-liquid ratio of the aconite granules to pure water is 1:8 - 12, and the ultrasonic power is 400 - 600 W.

[0113] S3. When the water temperature reaches a constant temperature of 70 - 80 °C, stir the aconite granules again for 4 hours, and then take the filtrate (i.e., the extract). The solid-liquid ratio of the aconite granules to pure water is 1:8 - 12.

[0114] S4. After filtering the extract twice, boil it for at least 1 hour. Use a tubular centrifuge to centrifuge the filtrate at a speed of 19000 rpm / min at room temperature, and concentrate the medicinal liquid under vacuum according to a concentration ratio of 13:1.

[0115] S5. Add 95% ethanol to the concentrated medicinal liquid with a volume 4 - 6 times that of the concentrated liquid, so that the alcohol content of the alcohol precipitation solution reaches more than 80%, and carry out alcohol precipitation at a constant temperature of 4 °C for 8 - 10 hours.

[0116] S6. Resuspend the precipitate with absolute ethanol and centrifuge it at a speed of 3800 r / min for 15 minutes.

[0117] S7. Repeat step S6 twice.

[0118] S8. Filter and vacuum-dry the precipitate after the treatment in step S7 to obtain the crude polysaccharide.

[0119] S9. Weigh a certain mass of the crude polysaccharide and completely dissolve it with sterile injection water. Use a membrane filtration system to separate and purify the polysaccharide with a molecular weight of 3000 - 100000 Da.

[0120] S10. Pass the polysaccharide solution through an affinity purification column filled with a medium of coupled polysaccharide nucleic acid aptamer to specifically capture the polysaccharide molecules, accurately, efficiently, and thoroughly remove the impurities (including aconitine, proteins, and cellulose, etc.) in the polysaccharide solution. By changing the pH of the affinity purification column, change the conformation of the polysaccharide nucleic acid aptamer, and obtain high-purity aconite polysaccharide after elution and freeze-drying.

[0121] Figure 3 This is the glucose standard curve graph prepared by the phenol-sulfuric acid method of the present invention. The elution product is detected by an ultraviolet spectrophotometer and a high-performance liquid chromatograph. The detection results of the ultraviolet spectrophotometer are shown in Table 1, and the chromatogram detection results are shown in Figure 4. Among them, Figure 4-1 This is the result graph of the total aconitine content of the crude aconite polysaccharide (Example 1), and the peak table is shown in Table 2. Figure 4-2 This is the result graph of the total aconitine content of the high-purity aconite polysaccharide (Example 3) without purification using nucleic acid aptamer, and the peak table is shown in Table 3. Figure 4-3This is a high-purity aconite polysaccharide purified using nucleic acid aptamers (Example 4) using a mixed standard of total aconitine (source: China Food and Drug Inspection Institute, batch number 112029-202302).

[0122]

[0123] Table 1

[0124]

[0125] Table 2

[0126]

[0127] Table 3

[0128] Note: "0%" in the table means that the content of aconitine in the sample does not exceed the detection limit of the instrument.

[0129] It can be seen that the use of a membrane filtration system and a polysaccharide aptamer affinity purification column in the crude polysaccharide of Radix Aconiti Lateralis Preparata increased the polysaccharide content of Radix Aconiti Lateralis Preparata to 99.44%.

[0130] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing high-purity aconite polysaccharide, characterized in that, It includes: S1. Dry and crush radix aconiti lateralis preparata to obtain aconite granules; S2. Perform ultrasonic stirring on the aconite granules and water, and filter to obtain the first filtrate; S3. Heat and stir the aconite granules and water after step S2, and filter to obtain the second filtrate; S4. Boil, centrifuge, and concentrate the first filtrate and the second filtrate to obtain a concentrated solution; S5. Add ethanol to the concentrated solution, and obtain an ethanol precipitation precipitate after alcohol precipitation; S6. Resuspend and centrifuge the ethanol precipitation precipitate with absolute ethanol for multiple times (the number of times is greater than or equal to 2) to obtain a resuspended precipitate; S7. Filter the resuspended precipitate by suction and perform vacuum drying to obtain crude aconite polysaccharide; S8. Completely dissolve the crude aconite polysaccharide in water, and separate the polysaccharide using a membrane filtration system to obtain an aconite polysaccharide solution; S9. Pass the aconite polysaccharide solution through an affinity purification column of aconite polysaccharide nucleic acid aptamer to remove impurities in the aconite polysaccharide solution, and then perform freeze-drying to obtain high-purity aconite polysaccharide.

2. The preparation method according to claim 1, characterized in that, In the step S1, the particle size of the aconite granules is 15 - 20 mesh.

3. The preparation method according to claim 1, wherein, In the step S2, the solid-liquid ratio of the aconite granules to water is 1:8 - 12, the ultrasonic power is 400 - 600W, and the ultrasonic time is 8 - 12 hours.

4. The preparation method according to claim 1, wherein In the step S3, the heating temperature is 70 - 80°C, the stirring time is 4 - 6 hours, and the solid-liquid ratio of the aconite granules and water after step S2 is 1:8 - 12.

5. The preparation method according to claim 1, characterized in that, In the step S4, the boiling time is greater than or equal to 1 hour, the boiling temperature is 90 - 110°C, the centrifugation speed is 15000 - 20000 rpm / minute, the centrifugation time is 10 - 20 minutes, and the volume ratio before and after concentration is 11 - 14:

1.

6. The preparation method according to claim 1, wherein In the step S5, the alcohol content of the ethanol is greater than or equal to 80%, the alcohol precipitation time is 8 - 10 hours, and the alcohol precipitation temperature is 2 - 4°C; In the step S6, the centrifugation speed is 3500 - 4000 rpm / minute, and the centrifugation time is 10 - 20 minutes.

7. The preparation method according to claim 1, characterized in that, In the step S8, the filler of the affinity purification column of aconite polysaccharide nucleic acid aptamer is obtained by covalently coupling an aptamer with the nucleic acid sequence such as SEQ ID NO.1 to a carrier of polyethylene glycol-modified agarose.

8. The preparation method according to claim 7, characterized in that, The preparation steps of the aconite polysaccharide aptamer affinity purification column include: A1. Prepare polyethylene glycol-modified agarose, and then perform swelling and washing using polyethylene glycol-modified agarose as a carrier; A2. Prepare an amino-modified aptamer, and then renature the amino-modified aptamer; A3. Couple, block, wash, and pack the polyethylene glycol-modified agarose treated in step A1 with the amino-modified aptamer treated in step A2 to obtain an aconite polysaccharide aptamer affinity purification column.

9. The preparation method according to claim 1, characterized in that, In the step S9, the membrane filtration system separates and purifies polysaccharides with a molecular weight of 3000 - 100000 Da.

10. A high-purity aconite polysaccharide, characterized in that, The high-purity aconite polysaccharide is prepared by the preparation method described in any one of claims 1 - 9.

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