A purification method for simultaneously obtaining two high-purity cobra analgesic peptides

Abstract

The present invention discloses a purification method for simultaneously obtaining two high-purity cobra analgesic peptides, and belongs to the field of biomedical technology. The purification method comprises dissolving cobra venom lyophilized powder in Tris-HCl buffer, and purifying it successively through CM-Sepharose cation exchange column and size exclusion chromatography column, wherein the CM-Sepharose cation exchange column is balanced with Tris-HCl buffer before loading, and the pH value of Tris-HCl buffer is 7.3-7.7. The present invention is purified only by two-step column chromatography steps of CM-Sepharose cation exchange column and size exclusion chromatography column, and no organic solvent is used in the purification process, and no high concentration salt is used, and all operations are performed at room temperature or below, which can maximize the biological activity of cobra analgesic peptide.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a purification method for simultaneously obtaining two high-purity cobra analgesic peptides. Background Art

[0002] Biotoxins are a class of biologically significant natural chemical products with a wide range of practical applications. As an important source of traditional toxins, some biotoxins have been used as effective clinical drugs and actively promoted drug innovation; on the other hand, many biotoxins can be used as lead compounds for innovative drugs, playing an irreplaceable role in drug screening, molecular design and the discovery of new drug targets.

[0003] As an important component of biological toxins, the composition of snake venom is very complex, mainly including neurotoxins (NT), cytotoxins (CTXs), phospholipase A2 (PLA2), bioactive peptides and other small molecules. Snake venom components have multiple biological activities, including anti-inflammatory, anti-tumor, hemostasis / anticoagulant, thrombolysis, analgesia, etc.

[0004] Neurotoxins are important active components in snake venoms, mainly distributed in the venom of rattlesnakes in the cobra family, sea snake family and viper family. In ancient my country, there was a folk remedy of using cobra to soak wine and drink to treat low back and leg pain. Cobra venom neurotoxins have a central analgesic effect similar to morphine, and are used for analgesic treatment of intractable pain, malignant tumor pain and joint pain. The analgesic effect of neurotoxins is different from that of opioids, and is characterized by high potency, long duration, no tolerance and addiction. It is a very promising new analgesic. Cobratide is a neurotoxin extracted from the venom of the Chinese cobra. It has a strong analgesic effect, long duration and high safety factor. As a biochemical drug of natural origin, it has the characteristics of good analgesic effect, small side effects, and no drug tolerance and dependence.

[0005] Studies have shown that the content of neurotoxins is higher in cobra venom, and a variety of neurotoxins have been isolated from cobra venom, among which short-chain neurotoxins composed of 60 to 62 amino acids have better analgesic effects. Drugs such as Ketongning and Keluoqu developed using short-chain neurotoxins compete with acetylcholine for acetylcholine receptors (nAChR) at the neuromuscular junction, blocking the nerve signals transmitted by acetylcholine to achieve the effect of analgesia. It is mainly used clinically to treat trigeminal neuralgia, sciatica, migraine, herpes pain, etc., with good clinical effects. In addition to its analgesic effect, it has good therapeutic effects in anti-rheumatic, anti-tumor and nervous system diseases. Therefore, the separation and purification of short-chain neurotoxins from cobra venom is one of the important ways to obtain analgesics.

[0006] At present, the separation and extraction of cobra neurotoxins from complex cobra snake venom mainly uses salting out, gel filtration chromatography, affinity chromatography, ion exchange chromatography and other methods. Among them, salting out requires a process of protein denaturation and renaturation, which results in serious loss of active protein and can only achieve the purpose of coarse separation; gel filtration chromatography can separate substances with different molecular weights using molecular sieves, but it is difficult to separate polypeptides or proteins with small molecular weight differences; affinity chromatography fillers are difficult to prepare, expensive, and have harsh conditions for use; ion exchange chromatography effectively separates target proteins based on different isoelectric points. Therefore, it is necessary to combine two or more separation methods to establish a stable, effective and reproducible separation method to extract high-purity neurotoxins and cobot peptides from cobra venom.

[0007] The national drug standard for cobotide (WS1-XG-009-2000) requires that the relative percentage of the main peak in the HPLC purity test should not be less than 90.0%. The content and purity of pharmaceutical raw materials play a decisive role in ensuring the quality, efficacy, safety, and dosage form of drugs. The existing extraction methods of cobra neurotoxin and cobotide may have problems such as low utilization rate of snake venom raw materials, relatively low product purity, and unstable content and efficacy between raw material batches.

[0008] Therefore, it is necessary to develop a stable, reliable, easy-to-operate, time-saving and labor-saving purification method to obtain high-purity analgesic neurotoxin from cobra venom.

[0009] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0010] The purpose of the present invention is to solve the problems existing in the existing cobra neurotoxin extraction methods, such as low utilization rate of snake venom raw materials, relatively low product purity, unstable content and efficacy between raw material batches, and to provide a stable and reliable purification method with high extraction product purity for simultaneously obtaining two high-purity cobra analgesic peptides.

[0011] The first aspect of the present invention provides a purification method for simultaneously obtaining two high-purity cobra analgesic peptides, comprising the following steps:

[0012] The cobra venom freeze-dried powder is dissolved in Tris-HCl buffer and purified by CM-Sepharose cation exchange column and size exclusion chromatography column in sequence, wherein the CM-Sepharose cation exchange column is balanced with Tris-HCl buffer before loading, and the pH value of the Tris-HCl buffer is 7.3-7.7.

[0013] In one embodiment of the present invention, the pH value of the Tris-HCl buffer is 7.5.

[0014] In one embodiment of the present invention, the size exclusion chromatography column is a TSKgel G2000SW size exclusion chromatography column.

[0015] In one embodiment of the present invention, the purification method comprises the following steps:

[0016] 1) Take lyophilized powder of cobra venom, dissolve it in Tris-HCl buffer, centrifuge at 10000 rpm at 4°C for 5-15 min, and take the supernatant for later use;

[0017] 2) After equilibration of the CM-Sepharose cation exchange column with Tris-HCl buffer, the supernatant obtained in step 1) was loaded onto the column, and gradient elution was performed with 280 nm as the detection wavelength to collect the P9 fraction and the P11 fraction with analgesic activity respectively;

[0018] 3) The collected fractions were dialyzed at 4°C overnight and then freeze-dried to obtain crude cobra analgesic peptides P9 and P11, respectively;

[0019] 4) Take the crude products of cobra analgesic peptides P9 and P11 respectively, dissolve them in NH4OAc aqueous solution, centrifuge at 10000 rpm at 4°C for 5-15 minutes, and take the supernatant for later use;

[0020] 5) equilibrate a TSKgel G2000SW size exclusion chromatography column with an NH4OAc aqueous solution, load the supernatant of the cobra analgesic peptide P9 obtained in step 4), elute with an NH4OAc aqueous solution, use 280 nm as the detection wavelength, and collect the size exclusion fraction No. 4 containing the cobra analgesic peptide P9;

[0021] 6) Continue to load the supernatant of cobra analgesic peptide P11 obtained in step 4) onto a TSKgel G2000SW size exclusion chromatography column, elute with NH4OAc aqueous solution, use 280 nm as the detection wavelength, and collect the size exclusion fraction No. 4 containing cobra analgesic peptide P11;

[0022] 7) The fractions collected in steps 5) and 6) were dialyzed overnight at 4° C. using dialysis bags and then freeze-dried to obtain pure cobra analgesic peptides P9 and P11.

[0023] In one embodiment of the present invention, the gradient elution in step 2) adopts linear gradient elution of NaCl aqueous solution, the minimum concentration of the NaCl aqueous solution is 0M, and the maximum concentration of the NaCl aqueous solution is 0.8-1M.

[0024] In one embodiment of the present invention, the dialysis uses a dialysis bag with a molecular weight cut-off of 500 to 2500 Da.

[0025] In one embodiment of the present invention, the dialysis uses a dialysis bag with a molecular weight cut-off of 1000 Da.

[0026] In one embodiment of the present invention, the concentration of the NH4OAc aqueous solution is 0.05-0.2M.

[0027] In one embodiment of the present invention, the concentration of the NH4OAc aqueous solution is 0.1M.

[0028] In one embodiment of the present invention, the purification method comprises the following steps:

[0029] 1) Weigh 2 g of cobra venom freeze-dried powder, dissolve in 10 mL of 0.2 M Tris-HCl buffer, centrifuge at 10,000 rpm for 10 min at 4 °C, and take the supernatant for later use;

[0030] 2) After equilibration of the CM-Sepharose cation exchange column with 0.2 M Tris-HCl buffer, the supernatant obtained in step 1) was loaded onto the column, and eluted with a linear gradient of 0 to 0.8 M NaCl, with 280 nm as the detection wavelength, to collect the P9 fraction and the P11 fraction with analgesic activity, respectively;

[0031] 3) The collected fractions were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain crude cobra analgesic peptides P9 and P11, respectively;

[0032] 4) Weigh 30 mg of crude cobra analgesic peptides P9 and P11, respectively, dissolve in 1 mL of 0.1 M NH4OAc aqueous solution, centrifuge at 10000 rpm for 10 min at 4°C, and take the supernatant for later use;

[0033] 5) equilibrate a TSKgel G2000SW size exclusion chromatography column with a 0.1M NH4OAc aqueous solution, load the supernatant of the cobra analgesic peptide P9 obtained in step 4), elute with a 0.1M NH4OAc aqueous solution, use 280nm as the detection wavelength, and collect the size exclusion fraction No. 4 containing the cobra analgesic peptide P9;

[0034] 6) Continue to take the supernatant of cobra analgesic peptide P11 obtained in step 4) and load it onto a TSKgel G2000SW size exclusion chromatography column, elute with 0.1M NH4OAc aqueous solution, use 280nm as the detection wavelength, and collect the size exclusion fraction No. 4 containing cobra analgesic peptide P11;

[0035] 7) The fractions collected in steps 5) and 6) were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain pure cobra analgesic peptides P9 and P11.

[0036] Compared with the prior art, the technical effects achieved by the present invention are as follows:

[0037] (1) The present invention only uses two column chromatography steps of CM-Sepharose cation exchange column and size exclusion chromatography column for purification. No organic solvent or high concentration salt is used in the purification process. All operations are performed at room temperature or below, which can maximize the biological activity of cobra analgesic peptide.

[0038] (2) The present invention uses a Tris-HCl buffer in a specific pH range in combination with a specific NaCl salt gradient range, and uses a CM-Sepharose cation exchange column to separate and purify the cobra venom lyophilized powder, which can improve the purification efficiency and purity of the two cobra analgesic peptides;

[0039] (3) The present invention can simultaneously purify two cobra analgesic peptides through a set of purification processes, and the purified cobra analgesic peptides have high purity and stable quality, and the HPLC purity reaches more than 98%;

[0040] (4) The present invention has simple steps and a large sample loading capacity, which is beneficial to the purification process control and improving the degree of automation, and is conducive to large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a cation exchange chromatography spectrum according to Example 1 of the present invention;

[0042] Figure 2 is the size exclusion chromatogram of P9 in Example 1 of the present invention;

[0043] Figure 3 is the size exclusion chromatogram of P11 in Example 1 of the present invention;

[0044] Figure 4 This is the HPLC spectrum of the cobra analgesic peptide P9 obtained according to Example 1 of the present invention;

[0045] Figure 5 This is the HPLC spectrum of the cobra analgesic peptide P11 obtained according to Example 1 of the present invention;

[0046] Figure 6 This is a high-resolution mass spectrum of cobra analgesic peptide P9 obtained according to Example 1 of the present invention;

[0047] Figure 7This is a high-resolution mass spectrum of the cobra analgesic peptide P11 obtained according to Example 1 of the present invention. DETAILED DESCRIPTION

[0048] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0049] The technical solution of the present invention is described below by specific embodiments. It should be understood that one or more steps mentioned in the present invention do not exclude the existence of other methods and steps before and after the combination step, or other methods and steps can be inserted between these explicitly mentioned steps. It should also be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Unless otherwise specified, the numbering of each method step is only for the purpose of identifying each method step, and does not limit the order of arrangement of each method or limit the scope of implementation of the present invention. The change or adjustment of the relative relationship thereof can also be regarded as the scope of implementation of the present invention without substantial changes in the technical content.

[0050] The sources of the raw materials and instruments used in the examples are not particularly limited and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0051] Example 1

[0052] A purification method for simultaneously obtaining two high-purity cobra analgesic peptides comprises the following steps:

[0053] 1) Weigh 2 g of cobra venom freeze-dried powder, dissolve in 10 mL of 0.2 M Tris-HCl buffer (pH 7.5), centrifuge at 10,000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0054] 2) After equilibration of the CM-Sepharose cation exchange column with 0.2 M Tris-HCl buffer (pH 7.5), the supernatant obtained in step 1) was loaded onto the column and eluted with a linear gradient of 0-0.8 M NaCl aqueous solution, with 280 nm as the detection wavelength, and the P9 (650-680 mL) and P11 (770-820 mL) fractions having analgesic activity were collected respectively.

[0055] 3) The collected fractions were dialyzed overnight at 4°C using dialysis bags with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain crude cobra analgesic peptides P9 and P11, respectively.

[0056] 4) Weigh 30 mg of crude cobra analgesic peptides P9 and P11, respectively, dissolve in 1 mL of 0.1 M NH4OAc aqueous solution, centrifuge at 10000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0057] 5) Equilibrate a TSKgel G2000SW size exclusion chromatography column with a 0.1 M NH4OAc aqueous solution, load the supernatant of the cobra analgesic peptide P9 obtained in step 4), elute with a 0.1 M NH4OAc aqueous solution, use 280 nm as the detection wavelength, and collect the size exclusion fraction No. 4 (57-59 mL) containing the cobra analgesic peptide P9.

[0058] 6) Continue to take the supernatant of cobra analgesic peptide P11 obtained in step 4) and load it onto a TSKgel G2000SW size exclusion chromatography column, elute with 0.1M NH4OAc aqueous solution, use 280nm as the detection wavelength, and collect the size exclusion fraction No. 4 (56-58mL) containing cobra analgesic peptide P11.

[0059] 7) The fractions collected in steps 5) and 6) were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain pure cobra analgesic peptides P9 and P11.

[0060] HPLC analysis: Prepare a 1 mg / mL cobra analgesic peptide P9 solution with a mobile phase, inject 10 μL, and use a C18 column for HPLC analysis. The HPLC spectrum is as follows: Figure 4 As shown. After testing, the purity of cobra analgesic peptide P9 was 98.822%. A cobra analgesic peptide P11 solution with a concentration of 1 mg / mL was prepared with a mobile phase, 10 μL was injected, and a C18 column was selected for HPLC analysis. The HPLC spectrum is shown as follows Figure 5 As shown in the figure, the purity of the cobra analgesic peptide P11 was 100%.

[0061] Mass spectrometry analysis: A 1 mg / mL solution of cobra analgesic peptide P9 was prepared with the mobile phase, and after dilution, it was analyzed using ESI high-resolution mass spectrometry. The high-resolution mass spectrum is shown in Figure 6 As shown. After testing, the molecular weight of cobra analgesic peptide P9 is 6808.0424Da. A cobra analgesic peptide P11 solution with a concentration of 1 mg / mL was prepared with the mobile phase, and after dilution, it was analyzed using ESI high-resolution mass spectrometry. The high-resolution mass spectrum is shown in Figure 7 As shown in Figure 2, the molecular weight of the cobra analgesic peptide P11 was found to be 6959.0075 Da.

[0062] This method uses ion exchange with a large sample loading capacity per gram of filler as the first purification step, which is beneficial to expanding the purification flux and can effectively separate polypeptide protein toxins with different isoelectric points. Two cobra analgesic peptides can be obtained by the same process; the second step is size exclusion chromatography to remove impurities of different molecular weights to obtain a high-purity target product.

[0063] Example 2

[0064] A purification method for simultaneously obtaining two high-purity cobra analgesic peptides comprises the following steps:

[0065] 1) Weigh 2 g of cobra venom freeze-dried powder, dissolve in 10 mL of 0.2 M Tris-HCl buffer (pH 7.3), centrifuge at 10,000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0066] 2) After equilibration of the CM-Sepharose cation exchange column with 0.2 M Tris-HCl buffer (pH 7.3), the supernatant obtained in step 1) was loaded onto the column and eluted with a linear gradient of 0-0.8 M NaCl aqueous solution, with 280 nm as the detection wavelength, and the P9 (675-705 mL) and P11 (795-845 mL) fractions having analgesic activity were collected respectively.

[0067] 3) The collected fractions were dialyzed overnight at 4°C using dialysis bags with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain crude cobra analgesic peptides P9 and P11, respectively.

[0068] 4) Weigh 30 mg of crude cobra analgesic peptides P9 and P11, respectively, dissolve in 1 mL of 0.1 M NH4OAc aqueous solution, centrifuge at 10000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0069] 5) Equilibrate a TSKgel G2000SW size exclusion chromatography column with a 0.1 M NH4OAc aqueous solution, load the supernatant of the cobra analgesic peptide P9 obtained in step 4), elute with a 0.1 M NH4OAc aqueous solution, use 280 nm as the detection wavelength, and collect the size exclusion fraction No. 4 (58-60 mL) containing the cobra analgesic peptide P9.

[0070] 6) Continue to take the supernatant of cobra analgesic peptide P11 obtained in step 4) and load it onto a TSKgel G2000SW size exclusion chromatography column, elute with 0.1M NH4OAc aqueous solution, use 280nm as the detection wavelength, and collect the size exclusion 4 fraction (55-57mL) containing cobra analgesic peptide P11.

[0071] 7) The fractions collected in steps 5) and 6) were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain pure cobra analgesic peptides P9 and P11.

[0072] HPLC analysis: C18 chromatographic column was used to perform HPLC analysis on P9 and P11. The purity of P9 and P11 was 98.035% and 98.348%, respectively.

[0073] Example 3

[0074] A purification method for simultaneously obtaining two high-purity cobra analgesic peptides comprises the following steps:

[0075] 1) Weigh 2 g of cobra venom freeze-dried powder, dissolve in 10 mL of 0.2 M Tris-HCl buffer (pH 7.7), centrifuge at 10,000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0076] 2) After equilibration of the CM-Sepharose cation exchange column with 0.2 M Tris-HCl buffer (pH 7.7), the supernatant obtained in step 1) was loaded onto the column and eluted with a linear gradient of 0-0.8 M NaCl aqueous solution, with 280 nm as the detection wavelength, and the P9 (640-670 mL) and P11 (760-810 mL) fractions having analgesic activity were collected respectively.

[0077] 3) The collected fractions were dialyzed overnight at 4°C using dialysis bags with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain crude cobra analgesic peptides P9 and P11, respectively.

[0078] 4) Weigh 30 mg of crude cobra analgesic peptides P9 and P11, respectively, dissolve in 1 mL of 0.1 M NH4OAc aqueous solution, centrifuge at 10000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0079] 5) Equilibrate a TSKgel G2000SW size exclusion chromatography column with a 0.1 M NH4OAc aqueous solution, load the supernatant of the cobra analgesic peptide P9 obtained in step 4), elute with a 0.1 M NH4OAc aqueous solution, use 280 nm as the detection wavelength, and collect the size exclusion fraction No. 4 (57-59 mL) containing the cobra analgesic peptide P9.

[0080] 6) Continue to take the supernatant of cobra analgesic peptide P11 obtained in step 4) and load it onto a TSKgel G2000SW size exclusion chromatography column, elute with 0.1M NH4OAc aqueous solution, use 280nm as the detection wavelength, and collect the size exclusion 4 fraction (58-60mL) containing cobra analgesic peptide P11.

[0081] 7) The fractions collected in steps 5) and 6) were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain pure cobra analgesic peptides P9 and P11.

[0082] HPLC analysis: C18 column was used to perform HPLC analysis on P9 and P11. The purity of P9 and P11 was 98.313% and 99.002%, respectively.

[0083] Example 4

[0084] A purification method for simultaneously obtaining two high-purity cobra analgesic peptides comprises the following steps:

[0085] 1) Weigh 2 g of cobra venom freeze-dried powder, dissolve in 10 mL of 0.2 M Tris-HCl buffer (pH 7.5), centrifuge at 10,000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0086] 2) After equilibration of the CM-Sepharose cation exchange column with 0.2 M Tris-HCl buffer (pH 7.5), the supernatant obtained in step 1) was loaded onto the column and eluted with a linear gradient of 0 to 1 M NaCl aqueous solution, with 280 nm as the detection wavelength, and the P9 (650 to 680 mL) and P11 (770 to 820 mL) fractions having analgesic activity were collected respectively.

[0087] 3) The collected fractions were dialyzed overnight at 4°C using dialysis bags with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain crude cobra analgesic peptides P9 and P11, respectively.

[0088] 4) Weigh 30 mg of crude cobra analgesic peptides P9 and P11, respectively, dissolve in 1 mL of 0.1 M NH4OAc aqueous solution, centrifuge at 10000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0089] 5) Equilibrate a TSKgel G2000SW size exclusion chromatography column with a 0.1 M NH4OAc aqueous solution, load the supernatant of the cobra analgesic peptide P9 obtained in step 4), elute with a 0.1 M NH4OAc aqueous solution, use 280 nm as the detection wavelength, and collect the size exclusion fraction No. 4 (57-59 mL) containing the cobra analgesic peptide P9.

[0090] 6) Continue to take the supernatant of cobra analgesic peptide P11 obtained in step 4) and load it onto a TSKgel G2000SW size exclusion chromatography column, elute with 0.1M NH4OAc aqueous solution, use 280nm as the detection wavelength, and collect the size exclusion fraction No. 4 (56-58mL) containing cobra analgesic peptide P11.

[0091] 7) The fractions collected in steps 5) and 6) were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain pure cobra analgesic peptides P9 and P11.

[0092] HPLC analysis: C18 column was used to perform HPLC analysis on P9 and P11. The purity of P9 and P11 was 98.056% and 98.183%, respectively.

[0093] Comparative Example 1

[0094] A method for purifying cobra analgesic peptide comprises the following steps:

[0095] 1) Weigh 2 g of cobra venom freeze-dried powder, dissolve in 10 mL of 0.2 M Tris-HCl buffer (pH 8.0), centrifuge at 10,000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0096] 2) After equilibration of the CM-Sepharose cation exchange column with 0.2 M Tris-HCl buffer (pH 8.0), the supernatant obtained in step 1) was loaded onto the column and eluted with a linear gradient of 0 to 0.8 M NaCl aqueous solution, with 280 nm as the detection wavelength, and the A7 (600 to 625 mL) fraction and the A9 (720 to 760 mL) fraction with analgesic activity were collected respectively.

[0097] 3) The collected fractions were dialyzed overnight at 4°C using dialysis bags with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain crude cobra analgesic peptides A7 and A9, respectively.

[0098] 4) Weigh 30 mg of crude cobra analgesic peptides A7 and A9 respectively, dissolve in 1 mL of 0.1 M NH4OAc aqueous solution, centrifuge at 10000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0099] 5) Equilibrate a TSKgel G2000SW size exclusion chromatography column with a 0.1 M NH4OAc aqueous solution, load the supernatant of cobra analgesic peptide A7 obtained in step 4), elute with a 0.1 M NH4OAc aqueous solution, use 280 nm as the detection wavelength, and collect the size exclusion fraction No. 4 (58-60 mL) containing cobra analgesic peptide A7.

[0100] 6) Continue to take the supernatant of cobra analgesic peptide A9 obtained in step 4) and load it onto a TSKgel G2000SW size exclusion chromatography column, elute with 0.1M NH4OAc aqueous solution, use 280nm as the detection wavelength, and collect the size exclusion 4 fraction (56-58mL) containing cobra analgesic peptide A9.

[0101] 7) The fractions collected in steps 5) and 6) were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain pure cobra analgesic peptides A7 and A9.

[0102] HPLC analysis: A7 and A9 were analyzed by HPLC using a C18 column. The purity of A7 and A9 was found to be 81.356% and 83.285%, respectively.

[0103] The purity of the analgesic active component obtained by purifying the cobra venom freeze-dried powder by CM-Sepharose ion exchange (pH 8.0) and then by size exclusion chromatography is less than 90%, which does not meet the drug purity requirements.

[0104] Comparative Example 2

[0105] A method for purifying cobra analgesic peptide comprises the following steps:

[0106] 1) Weigh 2 g of cobra venom freeze-dried powder, dissolve in 10 mL of 0.2 M Tris-HCl buffer (pH 7.0), centrifuge at 10,000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0107] 2) After equilibration of the CM-Sepharose cation exchange column with 0.2 M Tris-HCl buffer (pH 7.0), the supernatant obtained in step 1) was loaded onto the column and eluted with a linear gradient of 0-0.8 M NaCl aqueous solution, with 280 nm as the detection wavelength, and the B7 (700-725 mL) fraction and the B9 (830-870 mL) fraction with analgesic activity were collected respectively.

[0108] 3) The collected fractions were dialyzed overnight at 4°C using dialysis bags with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain crude cobra analgesic peptides B7 and B9, respectively.

[0109] 4) Weigh 30 mg of crude cobra analgesic peptides B7 and B9 respectively, dissolve in 1 mL of 0.1 M NH4OAc aqueous solution, centrifuge at 10000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0110] 5) Equilibrate a TSKgel G2000SW size exclusion chromatography column with a 0.1 M NH4OAc aqueous solution, load the supernatant of cobra analgesic peptide B7 obtained in step 4), elute with a 0.1 M NH4OAc aqueous solution, use 280 nm as the detection wavelength, and collect the size exclusion fraction No. 4 (59-61 mL) containing cobra analgesic peptide B7.

[0111] 6) Continue to take the supernatant of cobra analgesic peptide B9 obtained in step 4) and load it onto a TSKgel G2000SW size exclusion chromatography column, elute with 0.1M NH4OAc aqueous solution, use 280nm as the detection wavelength, and collect the size exclusion fraction No. 4 (57-59mL) containing cobra analgesic peptide B9.

[0112] 7) The fractions collected in steps 5) and 6) were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain pure cobra analgesic peptides B7 and B9.

[0113] HPLC analysis: C18 column was used to perform HPLC analysis on B7 and B9. The results showed that the purity of B7 and B9 was 93.459% and 94.587% respectively.

[0114] The purity of the analgesic active component obtained by purifying the cobra venom freeze-dried powder by CM-Sepharose ion exchange (pH 7.0) and then by size exclusion chromatography was less than 98%, and the purification effect was significantly worse than that of Example 1 of the present invention.

[0115] Comparative Example 3

[0116] A method for purifying cobra analgesic peptide comprises the following steps:

[0117] 1) Weigh 2 g of cobra venom freeze-dried powder, dissolve in 10 mL of 0.2 M Tris-HCl buffer (pH 7.5), centrifuge at 10,000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0118] 2) After equilibration of the CM-Sepharose cation exchange column with 0.2 M Tris-HCl buffer (pH 7.5), the supernatant obtained in step 1) was loaded onto the column and eluted with a linear gradient of 0-0.8 M NaCl aqueous solution, with 280 nm as the detection wavelength, and the P9 (650-680 mL) and P11 (770-820 mL) fractions having analgesic activity were collected respectively.

[0119] 3) The collected fractions were dialyzed overnight at 4°C using dialysis bags with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain crude cobra analgesic peptides P9 and P11, respectively.

[0120] 4) Weigh 30 mg of crude cobra analgesic peptides P9 and P11, respectively, dissolve in 1 mL of 0.1 M NH4OAc aqueous solution, centrifuge at 10000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0121] 5) Equilibrate a Sephadex G25 size exclusion chromatography column with a 0.1 M NH4OAc aqueous solution, load the supernatant of the cobra analgesic peptide P9 obtained in step 4), elute with a 0.1 M NH4OAc aqueous solution, use 280 nm as the detection wavelength, and collect the size exclusion fraction No. 1 (60-100 mL) containing the cobra analgesic peptide P9.

[0122] 6) Continue to take the supernatant of cobra analgesic peptide P11 obtained in step 4) and load it onto a TSKgel G2000SW size exclusion chromatography column, elute with 0.1M NH4OAc aqueous solution, use 280nm as the detection wavelength, and collect the size exclusion 1 fraction (60-100mL) containing cobra analgesic peptide P11.

[0123] 7) The fractions collected in steps 5) and 6) were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain cobra analgesic peptides P9 and P11.

[0124] HPLC analysis: C18 chromatographic column was used to perform HPLC analysis on P9 and P11. The purity of P9 and P11 was 91.338% and 93.276%, respectively.

[0125] The purity of the analgesic active component obtained by purifying the cobra venom freeze-dried powder by CM-Sepharose ion exchange and then by Sephadex G25 size exclusion chromatography was less than 98%, and the purification effect was obviously worse than that of Example 1 of the present invention.

[0126] Comparative Example 4

[0127] A method for purifying cobra analgesic peptide comprises the following steps:

[0128] 1) Weigh 0.07 g of freeze-dried cobra venom powder, dissolve it in 0.5 mL of 0.1 M NH4OAc aqueous solution, centrifuge at 10000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0129] 2) After equilibrating a TSKgel G2000SW size exclusion chromatography column with a 0.1 M NH4OAc aqueous solution, the supernatant obtained in step 1) was loaded onto the column and eluted with a 0.1 M NH4OAc aqueous solution in a constant gradient, with 280 nm as the detection wavelength.

[0130] 3) Collect the chromatographic peak No. 4 (55-65 mL fraction) with analgesic activity.

[0131] 4) Repeat steps 1) to 3) three times. Combine the collected chromatographic peaks, dialyze overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dry to obtain the crude analgesic active component of cobra.

[0132] 5) Weigh 115 mg of the crude analgesic active component of cobra, dissolve it in 2 mL of 0.2 M Tris-HCl buffer (pH 7.5), centrifuge at 10000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0133] 6) After equilibration of the CM-Sepharose cation exchange column with 0.2 M Tris-HCl buffer (pH 7.5), the supernatant obtained in step 1) was loaded onto the column and eluted with a linear gradient of 0 to 0.8 M NaCl aqueous solution, with 280 nm as the detection wavelength.

[0134] 7) Collect the chromatographic peaks of C5 (655-670 mL fraction) and C6 (770-800 mL fraction) with analgesic activity respectively.

[0135] 8) The collected chromatographic peaks were dialyzed overnight at 4° C. using dialysis bags with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain freeze-dried powders of C5 and C6, respectively.

[0136] HPLC analysis: C18 column was used to perform HPLC analysis on C5 and C6. The results showed that the purity of C5 and C6 were 92.843% and 91.532%, respectively.

[0137] The sample loading amount that size exclusion chromatography can carry is small, and the resolution is not high. The purity of the obtained analgesic active component after cation exchange purification is still less than 98%, and the purification effect is obviously worse than that of Example 1 of the present invention.

[0138] Comparative Example 5

[0139] A method for purifying cobra analgesic peptide comprises the following steps:

[0140] 1) Weigh 2.0 g of cobra venom freeze-dried powder, dissolve in 50 mL of 0.15 M PB buffer (pH 7.5), centrifuge at 10,000 rpm for 10 min at 4°C, take the supernatant, add ammonium sulfate to 60% saturation for salting out, precipitate overnight at 4°C, centrifuge at 10,000 rpm for 15 min, and take the supernatant for later use.

[0141] 2) dialyzing overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000Da, and then freeze-drying to obtain a crude product of the analgesic active component of cobra.

[0142] 3) Weigh 1000 mg of the crude analgesic active component of cobra, dissolve it in 0.2 M Tris-HCl buffer (pH 7.5), centrifuge it at 10000 rpm for 10 min at 4°C, and take the supernatant for later use.

[0143] 4) After equilibration of the CM-Sepharose ion exchange column with 0.2 M Tris-HCl buffer (pH 7.5), the supernatant obtained in step 1) was loaded onto the column and eluted with a linear gradient of 0 to 0.8 M NaCl aqueous solution, with 280 nm as the detection wavelength.

[0144] 5) The chromatographic peaks of D7 (650-675 L fraction) and D8 (765-805 mL fraction) with analgesic activity were collected separately.

[0145] 6) The collected chromatographic peaks were dialyzed overnight at 4° C. using dialysis bags with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain freeze-dried powders of D7 and D8, respectively.

[0146] HPLC analysis: C18 column was used to perform HPLC analysis on B3 and B4. The results showed that the purity of D7 and D8 were 88.435% and 89.258%, respectively.

[0147] The purity of the analgesic active component obtained after salting out and purification by CM-Sepharose ion exchange of the cobra venom freeze-dried powder is less than 90%, which does not meet the drug purity requirements.

[0148] Table 1 Comparison of cobra venom purification results

[0149]

[0150] Experimental example

[0151] Acetic acid writhing analgesia test in mice: 56 SPF male mice, 18-22g, were selected and randomly divided into 4 experimental groups and 1 control group according to body weight, with 10-14 mice in each group. The mice in the control group were given 0.9% sodium chloride injection by intramuscular injection, and the mice in each experimental group were given 0.9% sodium chloride injection of cobra analgesic peptides P9 and P11 by intramuscular injection according to Table 2 (cobra analgesic peptides P9 and P11 were prepared according to the method of Example 1), and the administration volume was 2mL / kg. 1h after administration, 0.8% glacial acetic acid solution was injected into the abdominal cavity of each mouse, and the injection volume was 10mL / kg. The number of writhing times of the mice in different time periods after administration of 0.8% acetic acid solution was recorded (mouse abdomen concave, trunk and hind legs extended, buttocks raised, body twisted, etc.), 0-30min was the first stage writhing reaction (times / 30min), and 30-60min was the second stage writhing reaction (times / 30min).

[0152] The writhing inhibition rate of each dosage group was calculated according to the following formula. The results are shown in Table 2.

[0153] Torsion inhibition rate = (number of torsion in the control group - number of torsion in the drug-treated group) / number of torsion in the control group × 100%

[0154] Table 2 The writhing activity of each dose group of cobra analgesic peptide

[0155]

[0156] As can be seen from Table 2, the cobra analgesic peptides P9 and P11 had a significant inhibitory effect on the writhing of mice, which was significantly different from the control group, and the analgesic effect was significant.

[0157] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A purification method for simultaneously obtaining two high-purity cobra analgesic peptides, characterized in that: The purification method comprises the following steps: The cobra venom freeze-dried powder is dissolved in Tris-HCl buffer and purified by CM-Sepharose cation exchange column and size exclusion chromatography column in sequence, wherein the CM-Sepharose cation exchange column is balanced with Tris-HCl buffer before loading, and the pH value of the Tris-HCl buffer is 7.3-7.

7.

2. The purification method according to claim 1, characterized in that The pH value of the Tris-HCl buffer is 7.

5.

3. The method according to claim 1, characterized in that: The size exclusion chromatography column is a TSKgel G2000SW size exclusion chromatography column.

4. The purification method according to claim 1, characterized in that The purification method comprises the following steps: 1) Take lyophilized powder of cobra venom, dissolve it in Tris-HCl buffer, centrifuge at 10000 rpm at 4°C for 5-15 min, and take the supernatant for later use; 2) After equilibration of the CM-Sepharose cation exchange column with Tris-HCl buffer, the supernatant obtained in step 1) was loaded onto the column, and gradient elution was performed with 280 nm as the detection wavelength to collect the P9 fraction and the P11 fraction with analgesic activity respectively; 3) The collected fractions were dialyzed at 4°C overnight and then freeze-dried to obtain crude cobra analgesic peptides P9 and P11, respectively; 4) Take the crude products of cobra analgesic peptides P9 and P11 respectively, dissolve them in NH4OAc aqueous solution, centrifuge at 10000 rpm at 4°C for 5-15 minutes, and take the supernatant for later use; 5) equilibrate a TSKgel G2000SW size exclusion chromatography column with an NH4OAc aqueous solution, load the supernatant of the cobra analgesic peptide P9 obtained in step 4), elute with an NH4OAc aqueous solution, use 280 nm as the detection wavelength, and collect the size exclusion fraction No. 4 containing the cobra analgesic peptide P9; 6) Continue to load the supernatant of cobra analgesic peptide P11 obtained in step 4) onto a TSKgel G2000SW size exclusion chromatography column, elute with NH4OAc aqueous solution, use 280 nm as the detection wavelength, and collect the size exclusion fraction No. 4 containing cobra analgesic peptide P11; 7) The fractions collected in steps 5) and 6) were dialyzed overnight at 4° C. using dialysis bags and then freeze-dried to obtain pure cobra analgesic peptides P9 and P11.

5. The purification method according to claim 4, characterized in that The gradient elution in step 2) adopts linear gradient elution of NaCl aqueous solution, the lowest concentration of NaCl aqueous solution is 0M, and the highest concentration of NaCl aqueous solution is 0.8-1M.

6. The purification method according to claim 4, characterized in that The dialysis uses a dialysis bag with a molecular weight cut-off of 500 to 2500 Da.

7. The purification method according to claim 4, characterized in that The dialysis used a dialysis bag with a molecular weight cut-off of 1000 Da.

8. The purification method according to claim 4, characterized in that The concentration of the NH4OAc aqueous solution is 0.05-0.2M.

9. The purification method according to claim 4, characterized in that The concentration of the NH4OAc aqueous solution is 0.1M.

10. The purification method according to claim 1, characterized in that: The purification method comprises the following steps: 1) Weigh 2 g of cobra venom freeze-dried powder, dissolve in 10 mL of 0.2 M Tris-HCl buffer, centrifuge at 10,000 rpm for 10 min at 4 °C, and take the supernatant for later use; 2) After equilibration of the CM-Sepharose cation exchange column with 0.2 M Tris-HCl buffer, the supernatant obtained in step 1) was loaded onto the column, and eluted with a linear gradient of 0 to 0.8 M NaCl, with 280 nm as the detection wavelength, to collect the P9 fraction and the P11 fraction with analgesic activity, respectively; 3) The collected fractions were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain crude cobra analgesic peptides P9 and P11, respectively; 4) Weigh 30 mg of crude cobra analgesic peptides P9 and P11, respectively, dissolve in 1 mL of 0.1 M NH4OAc aqueous solution, centrifuge at 10000 rpm for 10 min at 4°C, and take the supernatant for later use; 5) equilibrate a TSKgel G2000SW size exclusion chromatography column with a 0.1M NH4OAc aqueous solution, load the supernatant of the cobra analgesic peptide P9 obtained in step 4), elute with a 0.1M NH4OAc aqueous solution, use 280nm as the detection wavelength, and collect the size exclusion fraction No. 4 containing the cobra analgesic peptide P9; 6) Continue to take the supernatant of cobra analgesic peptide P11 obtained in step 4) and load it onto a TSKgel G2000SW size exclusion chromatography column, elute with 0.1M NH4OAc aqueous solution, use 280nm as the detection wavelength, and collect the size exclusion fraction No. 4 containing cobra analgesic peptide P11; 7) The fractions collected in steps 5) and 6) were dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 1000 Da, and then freeze-dried to obtain pure cobra analgesic peptides P9 and P11.

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