High-purity aconiti lateralis radix polysaccharide and preparation method thereof

Abstract

The application provides a high-purity aconite polysaccharide and a preparation method thereof. The preparation method comprises the following steps: drying, crushing, ultrasonic stirring, heating stirring, boiling, centrifugation, concentration, alcohol precipitation, resuspension, centrifugation, suction filtration and vacuum drying of Bai Fupian to obtain a crude aconite polysaccharide; then completely dissolving the crude aconite polysaccharide in water, and separating polysaccharides by using a membrane filtration system to obtain an aconite polysaccharide solution; finally removing impurities in the aconite polysaccharide solution by passing through an aconite polysaccharide nucleic acid aptamer affinity purification column, and then freeze-drying to obtain the high-purity aconite polysaccharide. The ultrasonic stirring and heating stirring are used to improve the dissolution rate of the aconite polysaccharide; the boiling, concentration, alcohol precipitation, resuspension and freeze-drying are used to obtain the high-purity aconite polysaccharide crude product; then the membrane filtration system is used to screen specific aconite polysaccharides; finally, the aptamer is used to accurately and efficiently remove alkaloids, so that the purity of the aconite polysaccharide is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of detection of aconiti polysaccharide, and particularly relates to a high-purity aconiti polysaccharide and a preparation method thereof. BACKGROUND

[0002] Aconiti Radix first appeared in Shennong's Herbal Classic, and is a processed product of the subterranean root (lateral root) of Aconitum carmichaeli Debx., a perennial herbaceous plant of the Ranunculaceae family. At present, it is widely planted in Sichuan, Shaanxi, Guizhou and other regions of China. It is a traditional Chinese medicine with a very long history in China, and has the effects of restoring yang, tonifying fire and yang, and dispelling cold and relieving pain, and can be used for yang deficiency and cold limbs. The main chemical components of aconite 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, and only in recent years has the research on aconite polysaccharides gradually attracted people's attention.

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

[0004] At present, the preparation methods of aconite polysaccharide mainly include enzyme-assisted extraction, ultrasonic extraction, ultrasonic-assisted hot extraction, water immersion extraction, microwave extraction, and water extraction and alcohol precipitation extraction. However, the above methods have great differences in extraction methods, low extraction efficiency, alkaloid residues, and difficulty in polysaccharide purification in the preparation of aconite polysaccharide, resulting in great loss of polysaccharide, which leads to a polysaccharide yield of less than 50% by the above methods. Therefore, there is an urgent need for a method for improving the purity of aconite polysaccharide.

[0005] Nucleic acid aptamer has a wide range of applications, especially in the biomedical field, as a diagnostic tool, targeted drugs in clinical treatment, food and environmental monitoring, and vaccine development. Nucleic acid aptamer is a single-stranded nucleic acid molecule (DNA or RNA) selected from a large number of random sequences through a screening process, which can bind to a specific target molecule (such as protein, small molecule or cell). By constructing a single-stranded random oligonucleotide library, using the Systematic evolution of ligands by exponential enrichment (SELEX) for multiple enrichment and screening, in vitro selection of nucleic acid aptamer with high affinity to target can be achieved, thereby avoiding the difficulties caused by in vivo immune response. The working principle of aptamer affinity column is to use the selective adsorption of aptamer to the target molecule to realize the extraction and purification of the target molecule in the complex sample, and the adsorption is reversible. The purification of aptamer affinity column has become an important development direction. SUMMARY

[0006] The present application provides a high-purity aconite polysaccharide and a preparation method thereof to solve the problem that the polysaccharide yield extracted in the prior art is less than 50%.

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

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

[0009] S1, drying and crushing the Bai Fupian to obtain aconite particles;

[0010] S2, ultrasonic stirring treatment of aconite particles and water, and filtering to obtain a first filtrate;

[0011] S3, heating and stirring the aconite particles after S2 and water, and filtering to obtain a second filtrate;

[0012] S4, boiling, centrifuging and concentrating the first filtrate and the second filtrate to obtain a concentrated solution;

[0013] S5, adding ethanol to the concentrated solution, and obtaining an alcohol precipitation after alcohol precipitation;

[0014] S6, resuspending and centrifuging the alcohol precipitation multiple times using anhydrous ethanol to obtain a resuspended precipitate, the number of times being greater than or equal to 2;

[0015] S7, suction filtering the resuspended precipitate, and vacuum drying to obtain a crude aconite polysaccharide;

[0016] S8, completely dissolving the crude aconite polysaccharide with water, and separating the polysaccharide using a membrane filtration system to obtain an aconite polysaccharide solution;

[0017] S9, removing impurities in the aconite polysaccharide solution through an aconite polysaccharide nucleic acid aptamer affinity purification column, and then freeze-drying to obtain high-purity aconite polysaccharide.

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

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

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

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

[0022] In the S5 step, the alcohol degree 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 DEG C.

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

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

[0025] A1, preparing polyethylene glycol modified agarose, and then swelling and washing the polyethylene glycol modified agarose as a carrier;

[0026] A2, preparing amino-modified aptamer, and then renaturing the amino-modified aptamer;

[0027] A3, coupling, blocking, washing and column packing the polyethylene glycol modified agarose after the A1 step and the amino-modified aptamer after the A2 step to obtain the aconite polysaccharide aptamer affinity purification column.

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

[0029] The application also provides a high-purity aconite polysaccharide prepared by the above preparation method.

[0030] The beneficial effects of the present application are:

[0031] I. The present application improves the dissolution rate of aconite polysaccharide by ultrasonic stirring and heating stirring. Then, by boiling, concentrating, alcohol precipitation, resuspension, and freeze-drying, high-purity aconite polysaccharide crude product is obtained, and the temperature is controlled during the process to prevent high-temperature damage to the polysaccharide structure. Subsequently, the aconite polysaccharide with specific molecular weight and biological activity is screened by ultrafiltration. Finally, the purity of aconite polysaccharide is improved by using aptamer to accurately and efficiently remove alkaloids. The present application provides a wide application prospect for the development and utilization of aconite polysaccharide.

[0032] II. The application of nucleic acid aptamer in the screening of polysaccharide molecules involves the cooperation between chemistry, biochemistry, and molecular biology, aiming to better prepare high-purity aconite polysaccharide. The present application uses nucleic acid aptamer to capture polysaccharide and elute impurities by using nucleic acid aptamer affinity purification column, effectively solving the problems of alkaloid residue, low efficiency, and long time-consuming in aconite polysaccharide extract.

[0033] III. The present application uses aconite polysaccharide aptamer and polyethylene glycol modified agarose to prepare aconite polysaccharide aptamer affinity purification column, which is simple to prepare, low in price, and can be repeatedly used for more than 25 times.

[0034] IV. The present application has high purification efficiency of aconite polysaccharide, and the recovery rate of aconite polysaccharide is more than 99%. BRIEF DESCRIPTION OF DRAWINGS

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

[0036] Figure 2 is a schematic diagram of using coupling polysaccharide nucleic acid aptamer affinity purification column to purify polysaccharide;

[0037] Figure 3 is a glucose standard curve graph prepared by the phenol-sulfuric acid method of the present application;

[0038] Figure 4 is a chromatogram result of detecting the total aconitine (aconitine, hypaconitine, and mesaconitine) content in polysaccharide product by high-performance liquid chromatography of the present application; wherein, Figure 4-1 is the total aconitine result graph of detecting aconite polysaccharide crude product, Figure 4-2 is the total aconitine result graph of detecting high-purity aconite polysaccharide without using nucleic acid aptamer purification, Figure 4-3 is the total aconitine mixed standard graph of using nucleic acid aptamer purified high-purity aconite polysaccharide (source: China Food and Drug Inspection Research Institute, batch number 112029-202302).

[0039] Reference signs:

[0040] 1 - Inlet port plug, 2 - column, 3 - upper sieve plate, 4 - carrier packing, 5 - lower sieve plate, 6 - sample tube. DETAILED DESCRIPTION

[0041] The technical solutions of the present application are described below clearly and completely. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0042] The present application provides a preparation method of high-purity aconitum polysaccharide, comprising:

[0043] S1, drying and crushing white aconite tablets to obtain aconite particles.

[0044] In an embodiment, white aconite tablets are a kind of aconite, mainly produced in Sichuan, Shaanxi and other provinces.

[0045] In the S1 step, the white aconite tablets are crushed by using conventional crushing machines in the art. The particle size of the aconite particles is 15-20 mesh.

[0046] S2, ultrasonic stirring treatment of aconite particles and water, and filtering to obtain a first filtrate.

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

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

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

[0050] S3, heating and stirring of aconite particles after S2 step and water, and filtering to obtain a second filtrate.

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

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

[0053] S4, boiling, centrifuging and concentrating the first filtrate and the second filtrate to obtain a concentrated solution.

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

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

[0056] In the 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, adding ethanol to the concentrated solution, and obtaining an alcohol precipitation after alcohol precipitation.

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

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

[0060] S6, resuspending and centrifuging the alcohol precipitation multiple times using anhydrous ethanol to obtain a resuspended precipitate, the number of times being greater than or equal to 2.

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

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

[0063] S7, performing suction filtration on the resuspended precipitate to obtain a fuzi polysaccharide crude product by vacuum drying.

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

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

[0066] S8, the crude aconiti polysaccharide is completely dissolved in water, and a membrane filtration system is used to separate the polysaccharide to obtain an aconiti polysaccharide solution.

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

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

[0069] S9, the impurities in the aconiti polysaccharide solution are removed through an aconiti polysaccharide aptamer affinity purification column, and then freeze-drying is performed to obtain high-purity aconiti polysaccharide.

[0070] In the step S9, the impurities include aconitine, protein, cellulose and the like. The filler of the aconiti polysaccharide aptamer affinity purification column is polyethylene glycol modified agarose as a carrier, and then the nucleic acid aptamer with the nucleic acid sequence of SEQ ID NO. 1 is covalently coupled with the carrier to obtain the aconiti polysaccharide aptamer affinity purification column. Here, the polyethylene glycol modified agarose is covalently coupled with the amino modified aptamer, the coupling product is stable, and the coupling rate is high. The used nucleic acid aptamer is obtained by in vitro chemical synthesis method, which can ensure the correctness of the sequence and the consistency between batches, and greatly reduces the difference between different batches.

[0071] The present application makes full use of the advantages of high specificity and high affinity of the nucleic acid aptamer, and uses the aconiti polysaccharide aptamer to specifically bind to the aconiti polysaccharide in the sample, thereby greatly improving the purification efficiency of the affinity purification column. The nucleic acid aptamer of the present application is less affected by the operating environment and organic solvents, and is especially suitable for on-site purification of production lines in various industries.

[0072] Specifically, the preparation steps of the aconiti polysaccharide aptamer affinity purification column include: A1, preparing polyethylene glycol modified agarose, and then swelling and washing the polyethylene glycol modified agarose as a carrier; A2, preparing amino modified aptamer, and then renaturing the amino modified aptamer; A3, coupling, blocking, washing and column loading the polyethylene glycol modified agarose treated in the step A1 and the amino modified aptamer treated in the step A2 to obtain the aconiti polysaccharide aptamer affinity purification column.

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

[0074] Specifically, the step A1 comprises: A101 preparing polyethylene glycol modified agarose: agarose is mixed with equal volume of water, and is added into a reactor equipped with a pH electrode and a magnetic stirrer, 50-300 mg of polyethylene glycol is added into the agarose solution in an amount of 50-300 mg per mL of agarose solution, the pH value is adjusted to 11±0.1 by NaOH, the whole reaction pH value is controlled at 11±0.1, the temperature is controlled at 20℃±5℃, and the reaction is completed in 3-12 minutes; after the reaction is completed, equal volume of ice chips is quickly added into the above reaction solution, and is quickly poured into a Buchner funnel, and is washed by suction filtration with 10-15 times volume of cold buffer solution of the agarose solution, so that the hydroxyl groups on the surface are reacted with polyethylene glycol, and polyethylene glycol modified agarose is obtained; A102 swelling and washing of the carrier: 30-150 mg of carrier powder is soaked in 1-5 mL of 1 mM hydrochloric acid for 0.5-1 hour for swelling; the swollen carrier is washed with 1 mM hydrochloric acid for 3-6 times, the amount of hydrochloric acid is 2-5 mL each time, then is washed with distilled water for 2-5 times, the amount of distilled water is 1-5 mL each time, and finally is washed with Na2HPO4 buffer for 2-5 times, the amount of buffer is 1-5 mL each time.

[0075] Specifically, the step A2 comprises: A201 preparing amino-modified aptamer: a C7 indirect arm-(CH2)7- or a C6 indirect arm-(CH2)6- is connected to the 3' or 5' end of the nucleic acid aptamer through a covalent bond, and then an amino group is modified at the end of the C7 indirect arm or the C6 indirect arm through a covalent bond, to obtain an amino-modified aptamer; A202 renaturation of the amino-modified aptamer: 1-50D of the amino-modified aptamer is dissolved in 200-1000 μL of Na2HPO4 buffer, is renatured at 75-95℃ for 3-5 minutes, and is then placed at room temperature for 15-60 minutes.

[0076] Specifically, the A3 step comprises: A301 coupling: adding the amino-modified aptamer treated in A2 into the polyethylene glycol-modified agarose treated in A1 at 30°C and shaking overnight; A302 blocking: sequentially washing the coupling product obtained in A301 with 200 mM Na2HP04 aqueous solution at pH 8.0 for 2-5 times, each time with 15 mL, then washing with 0.1 M Tris-HCl buffer at pH 8.0 for 2-5 times, each time with 1-5 mL, then adding 0.1 M Tris-HCl solution at pH 8.0 for 2-5 mL, shaking at 30°C for 1-6 h to block the remaining active sites, and obtaining the carrier-aptamer coupling gel; A303 washing: sequentially washing the carrier-aptamer coupling gel obtained in A302 with acetic acid buffer and Tris-HCl buffer for 3-5 times, each time with 1-5 mL of acetic acid buffer or Tris-HCl buffer, and removing the uncoupled aptamer; and washing the coupling gel with 1-5 mL of binding buffer, and obtaining the coupling gel suspension for column loading; wherein the acetic acid buffer is 0.1 M acetic acid-sodium acetate aqueous solution containing 0.5 M NaCl at pH 4.0; the Tris-HCl buffer has a concentration of 0.1 M and contains 0.5 M NaCl at pH 8.0; the binding buffer contains 10 mM Tris, 120 mM NaCl, 5 mM KCl and 5 mM MgCl2 at pH 7.5; and A304 column loading: taking a solid-phase extraction column with a volume of 1-5 mL, loading the coupling gel suspension into the column until the gel height is 1 cm, adding 0.05 w / v% NaN3 solution for 0.5-3 mL, and storing at 4°C.

[0077] The use mode is as follows: 1) taking out the polysaccharide aptamer affinity purification column, opening the sample inlet plug, connecting the sample inlet with the syringe needle, and connecting the syringe to the pneumatic operation rack. 2) opening the sample outlet plug, equilibrating the affinity purification column with 5 mL of binding buffer, adjusting the air hole operation rack air pump pressure, and making the liquid flow out at a flow rate of 3 drops per second. 3) adding the above-mentioned redissolved solution into the affinity purification column, adjusting the flow rate to 1-2 drops per second, and flowing out the sample until the sample is completely flowed out of the affinity purification column. 4) washing the affinity purification column with 1 mL of binding buffer. 5) adding 1 mL of purified water, and collecting the elution product.

[0078] As Figure 2 The schematic diagram for purifying polysaccharide by using the coupling polysaccharide nucleic acid aptamer affinity purification column is shown in FIG. 1. The polysaccharide aptamer affinity purification column is used to extract the polysaccharide from the Fuzi.

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

[0080] In the present application, all the preparation raw materials are commercially available products well known to those skilled in the art, unless otherwise specified.

[0081] Example 1 crude aconite polysaccharide

[0082] S1, after drying the white aconite tablet, the dried white aconite tablet was crushed into granules by a crusher, and then the granules were passed through a 15-20 mesh sieve to obtain aconite granules.

[0083] S2, the aconite granules were weighed, and then the aconite granules were ultrasonically stirred with pure water at room temperature for 12 hours to obtain a filtrate (i.e. an extract). The solid-liquid ratio of the aconite granules to the pure water was 1:8-12, the ultrasonic power was 400-600 W, and the ultrasonic time was 8-12 hours.

[0084] S3, when the water temperature reached 70-80℃, the aconite granules were stirred again for 4 hours, and then a filtrate (i.e. an extract) was obtained. The solid-liquid ratio of the aconite granules to the pure water was 1:8-12.

[0085] S4, the extract was filtered twice and boiled for at least 1 hour. The filtrate was centrifuged at a speed of 19000 rpm / min using a tubular centrifuge at room temperature, and the filtrate was vacuum concentrated at a concentration ratio of 13:1.

[0086] S5, 95% ethanol was added to the concentrated filtrate at a volume of 4-6 times that of the concentrated filtrate, and the mixture was kept at a constant temperature of 4℃ for 8-10 hours.

[0087] S6, the precipitate was resuspended with anhydrous ethanol, and then centrifuged at a speed of 3800 r / min for 15 minutes.

[0088] S7, step S6 was repeated twice.

[0089] S8, the precipitate after step S7 was filtered and vacuum dried to obtain a crude polysaccharide.

[0090] Example 2 preparation of aconite polysaccharide aptamer

[0091] 1) Preparation of polyethylene glycol modified agarose: agarose was mixed with an equal volume of water, and then added to a reactor equipped with a pH electrode and a magnetic stirrer. Polyethylene glycol was added to the agarose solution at an amount of 50-300 mg per mL of agarose solution, and then the pH value was adjusted to 11±0.1 with NaOH. The whole reaction was controlled at a pH value of 11±0.1 and a temperature of 20℃±5℃. The reaction was completed in 3-12 minutes. After the reaction was completed, an equal volume of ice chips was quickly added to the reaction solution, and then quickly poured into a Buchner funnel. The surface hydroxyl groups were reacted with polyethylene glycol by washing with a cold buffer at a volume of 10-15 times that of the agarose solution, and thus polyethylene glycol modified agarose was obtained.

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

[0093] 3) Preparation of amino-modified aptamer: a C7 indirect arm -(CH2)7- or a C6 indirect arm -(CH2)6- was covalently linked to the 3' or 5' end of the nucleic acid aptamer, and then an amino group was modified at the end of the C7 indirect arm or the C6 indirect arm by a covalent bond to obtain an amino-modified aptamer.

[0094] 4) Aptamer renaturation: 1-5 OD of 3' or 5' amino-modified aptamer polysaccharide aptamer specific DNA was dissolved in 200-1000 μL of Na2HPO4 buffer, and was renatured at 75-95 °C for 3-5 minutes, and then was placed at room temperature for 15-60 minutes.

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

[0096] 6) Blocking: the coupling product obtained in step 5) was sequentially washed with 15 mL of 200 mM Na2HPO4 aqueous solution at pH 8.0 for 2-5 times, then was washed with 1-5 mL of 0.1 M Tris-HCl buffer at pH 8.0 for 2-5 times, then 2-5 mL of 0.1 M Tris-HCl solution at pH 8.0 was added, and was reacted at 30 °C on a shaker for 1-6 h to block the remaining active sites, thereby obtaining a carrier-aptamer coupling gel.

[0097] 7) Washing: the above carrier-aptamer coupling gel was alternately washed with acetic acid buffer and Tris-HCl buffer for 3-5 times, each time with 1-5 mL of acetic acid buffer or Tris-HCl buffer, to remove the uncoupled aptamer; the washed coupling gel was resuspended with 1-5 mL of binding buffer, and the obtained coupling gel suspension was prepared for column loading. The acetic acid buffer was 0.1 M acetic acid-sodium acetate aqueous solution containing 0.5 M NaCl, at pH 4.0; the Tris-HCl buffer had a concentration of 0.1 M and contained 0.5 M NaCl, at pH 8.0; the binding buffer contained 10 mM Tris, 120 mM NaCl, 5 mM KCl and 5 mM MgCl2, at pH 7.5.

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

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

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

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

[0102] S3, When the water temperature reaches 70-80°C constant temperature, stir the aconite granules again for 4 hours, and 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, 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 to the concentrated medicinal liquid, and the volume of ethanol is 4-6 times that of the concentrated liquid, so that the alcohol concentration of the alcohol precipitation solution reaches more than 80%, and constant temperature 4°C alcohol precipitation 8-10 hours.

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

[0106] S7, Repeat step S6 twice.

[0107] S8, After the precipitate treated in step S7 is filtered and vacuum dried, a crude polysaccharide is obtained.

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

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

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

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

[0112] S2, weigh the aconite particles, and use pure water to ultrasonically stir the aconite particles at room temperature for 12 hours, and then take the filtrate (i.e. the extract). The solid-liquid ratio of the aconite particles to pure water is 1:8-12, and the ultrasonic power is 400-600 W.

[0113] S3, when the water temperature reaches 70-80℃, the aconite particles are stirred again for 4 hours, and then the filtrate (i.e. the extract) is taken. The solid-liquid ratio of the aconite particles to pure water is 1:8-12.

[0114] S4, after boiling the extract for at least 1 hour, the filtrate is centrifuged at a speed of 19000 rpm / min using a tubular centrifuge at room temperature, and the filtrate is vacuum concentrated at a concentration ratio of 13:1.

[0115] S5, 95% ethanol is added to the concentrated filtrate at a volume of 4-6 times that of the concentrated solution, so that the alcohol concentration of the alcohol precipitation solution reaches more than 80%, and is kept at 4℃ for 8-10 hours.

[0116] S6, the precipitate is resuspended with anhydrous ethanol, and centrifuged at a speed of 3800 r / min for 15 minutes.

[0117] S7, step S6 is repeated twice.

[0118] S8, the precipitate treated in step S7 is suction filtered and vacuum dried to obtain a crude polysaccharide product.

[0119] S9, a certain amount of crude polysaccharide is completely dissolved with sterilized injection water, and a membrane filtration system is used to separate and purify polysaccharides with a molecular weight of 3000-100000 Da.

[0120] S10, the polysaccharide solution is passed through an affinity purification column filled with a coupling polysaccharide nucleic acid aptamer, and the polysaccharide molecules are specifically captured, and the impurities (including aconitine, protein and cellulose, etc.) in the polysaccharide solution are accurately and efficiently removed. The conformation of the polysaccharide nucleic acid aptamer is changed by changing the pH of the affinity purification column, and high-purity aconite polysaccharide is obtained after elution and freeze-drying.

[0121] Figure 3 is a glucose standard curve graph prepared by the phenol-sulfuric acid method. The eluted product is detected by ultraviolet spectrophotometry and high-performance liquid chromatography. The ultraviolet spectrophotometry detection results are shown in Table 1, and the chromatogram detection results are shown in Figure 4. Among them, Figure 4-1 is a total aconitine result graph for detecting crude aconite polysaccharide (Example 1), and the peak table is shown in Table 2, Figure 4-2 is a total aconitine result graph for detecting high-purity aconite polysaccharide (Example 3) without using nucleic acid aptamer purification, and the peak table is shown in Table 3, Figure 4-3is high purity aconite polysaccharide purified by using nucleic acid aptamer (Example 4) Total aconitine mixed standard chart (source: China Institute for Food and Drug Control, batch number 112029-202302) was used.

[0122]

[0123] Table 1

[0124]

[0125] Table 2

[0126]

[0127] Table 3

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

[0129] It can be seen that the crude aconite polysaccharide is improved by using membrane filtration system and polysaccharide aptamer affinity purification column, and the content of aconite polysaccharide reaches 99.44%.

[0130] Although the embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and alterations can be made thereto without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing high-purity Aconitum carmichaelii polysaccharide, characterized in that, include: S1. Dry and pulverize the white aconite root slices to obtain aconite granules; S2. The Aconitum carmichaelii granules and water are ultrasonically stirred and filtered to obtain the first filtrate. S3. Heat and stir the aconite granules and water after step S2, then filter to obtain the second filtrate. S4. Boil, centrifuge, and concentrate the first and second filtrates to obtain a concentrated solution; S5. Add ethanol to the concentrate, and after alcohol precipitation, obtain the alcohol precipitate. S6. The alcohol precipitate is resuspended and centrifuged multiple times with anhydrous ethanol to obtain a resuspended precipitate, with the number of resuspending times being greater than or equal to 2. S7. Filter the resuspended precipitate and dry it under vacuum to obtain crude Aconitum carmichaelii polysaccharide. S8. Dissolve the crude Aconitum carmichaelii polysaccharide completely in water and separate the polysaccharide using a membrane filtration system to obtain an Aconitum carmichaelii polysaccharide solution. S9. Impurities in the Aconitum carmichaelii solution are removed by using an affinity purification column for Aconitum carmichaelii nucleic acid aptamers, followed by freeze-drying to obtain high-purity Aconitum carmichaelii polysaccharide; wherein, the packing material of the affinity purification column for Aconitum carmichaelii nucleic acid aptamers is polyethylene glycol-modified agarose as a carrier, and then the nucleic acid aptamer with a nucleic acid sequence such as SEQ ID NO.1 is covalently coupled to the carrier.

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

3. The preparation method according to claim 1, characterized in that, In step S2, the solid-liquid ratio of Aconitum carmichaelii 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, characterized in that, In step S3, the heating temperature is 70-80℃, the stirring time is 4-6 hours, and the solid-liquid ratio of Aconitum carmichaelii granules to water after step S2 is 1:8-12.

5. The preparation method according to claim 1, characterized in that, In step S4, the boiling time is greater than or equal to 1 hour, the boiling temperature is 90-110℃, the centrifugation speed is 15000-20000 rpm, 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, characterized in that, In step S5, the alcohol content of ethanol is greater than or equal to 80%, the alcohol precipitation time is 8-10 hours, and the alcohol precipitation temperature is 2-4℃; in step S6, the centrifugation speed is 3500-4000 rpm, and the centrifugation time is 10-20 minutes.

7. The preparation method according to claim 1, characterized in that, The preparation steps of the Aconitum carmichaelii aptamer affinity purification column include: A1, preparing polyethylene glycol-modified agarose, and then using polyethylene glycol-modified agarose as a carrier for swelling and washing; A2, preparing amino-modified aptamers, and then refolding the amino-modified aptamers; A3, coupling, blocking, washing, and packing the polyethylene glycol-modified agarose treated in step A1 with the amino-modified aptamers treated in step A2 into an Aconitum carmichaelii aptamer affinity purification column.

8. The preparation method according to claim 1, characterized in that, The membrane filtration system separates and purifies polysaccharides with a molecular weight of 3,000-100,000 Da.

9. A high-purity Aconitum carmichaelii polysaccharide, characterized in that, The high-purity Aconitum carmichaelii polysaccharide is prepared by the preparation method according to any one of claims 1-8.

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