Preparation process of cobropeptide and snake venom substance A extracted from snake venom

Abstract

The application relates to the technical field of biological pharmacy, in particular to a preparation process of cobropeptide and snake venom substance A extracted from snake venom. The application adopts Proteomin POR15-S filler to separate the snake venom, and determines the corresponding separation method. The method can replace the existing filler to complete the separation of the snake venom, obtain the cobropeptide and the snake venom substance A, improve the utilization rate of the snake venom, and lay a foundation for the effective development and utilization of other substances of the snake venom. The finally obtained snake venom substance A has an antitumor effect, especially has the effects of inhibiting cervical cancer, nasopharyngeal carcinoma and gastric cancer, is relatively safe, and has the potential value of being developed into an antitumor drug, especially a cervical cancer, nasopharyngeal carcinoma and gastric cancer drug.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a preparation process for extracting cobopeptide and snake venom substance A from snake venom. Background Technology

[0002] Cobratide is a neurotoxin protein isolated and purified from the venom of the Chinese cobra. It is a polypeptide drug used for chronic pain such as late-stage cancer pain, chronic joint pain, and sciatica. Its primary structure consists of 62 amino acids with four groups of disulfide bonds, an isoelectric point of pH 8.8, and an amino acid sequence of: LECHNQQSSQ TPTTTGCSGG ETNCYKKRWRDHRGYRTERG CGCPSVKNGI EINCCTTDRC NN. Its molecular weight is 6944.9925 Da.

[0003] Cobopeptide accounts for about 6% of the dried venom powder from the Zhoushan cobra. About 94% of other substances in snake venom remain undeveloped and unutilized. Therefore, finding a method that can separate snake venom to obtain cobopeptide, as well as other effective substances from snake venom, is a current research focus. This research can improve the utilization rate of snake venom and lay the foundation for the effective development and utilization of other substances in snake venom. Summary of the Invention

[0004] To address the aforementioned technical problems in the existing technology, this invention provides a preparation process for extracting cobopeptide and snake venom substance A from snake venom, specifically achieved through the following technical solution:

[0005] A preparation process for extracting cobopeptide and snake venom substance A from snake venom is characterized by dissolving snake venom in 0.01M phosphate, using ion exchange chromatography with Proteomin POR15-S packing material, and using 0.01M phosphate buffer as mobile phase A and 1M NaCl-containing phosphate buffer as mobile phase B for elution and separation.

[0006] The venom in question is that of the Zhoushan cobra, specifically the Zhoushan cobra (Elapheidae family, Elaphe genus), and it originated from the Hongyun Snake Farm in Xiangyun County.

[0007] Furthermore, the following steps are included:

[0008] (1) Sample preparation: Take dried snake venom powder, dissolve it with 0.01M phosphate to prepare a 0.1-1g / ml solution, filter it, and take the filtrate for separation;

[0009] (2) Separation conditions: Ion exchange chromatography was used, with Proteomin POR15-S as the packing material, 0.01M phosphate buffer as mobile phase A and 1M NaCl phosphate buffer as mobile phase B for elution, the flow rate was 1-5 ml / min, and the detection wavelength was 280 nm.

[0010] (3) Separation and collection: Automatic collectors are used for collection, every 1 minute per sample. After separation, peaks are collected according to the spectrum.

[0011] (4) Add acetone to the separated and collected liquid, let it settle, filter and dry to obtain the final product.

[0012] Furthermore, the elution is gradient elution, and the elution gradient is as follows:

[0013] 0 100 0 25 95 5 75 90 10 100 0 100 101 100 0 125 100 0

[0014] Furthermore, the sample preparation involves dissolving dried snake venom powder in 0.01M phosphate to prepare a 0.5g / ml solution; the flow rate is 3ml / min.

[0015] Furthermore, the acetone is cold acetone at three times the volume of the drug solution; the cold precipitation is cold precipitation for 30 minutes; the filtration is vacuum filtration; the drying is low-temperature drying, specifically freeze drying, and the product is taken out when the vacuum degree of the freeze dryer is less than 1 Pa and the freeze drying time is greater than 24 hours.

[0016] Furthermore, in step (3), when a cross peak occurs during the collection process, the sample 1 minute before and after the peak bottom is discarded.

[0017] Furthermore, the method also includes a purification process: the crudely extracted cobopeptide or snake venom substance A is dissolved in water for injection to prepare a 0.1-1 g / ml solution. LH-20 packing material was eluted with water for injection at a flow rate of 2 ml / min and a detection wavelength of 280 nm for 70 min. The material was collected using an automatic collector at a rate of 1 min per packing material. After separation, peaks were collected according to the chromatogram and then freeze-dried to obtain the final product.

[0018] Snake venom substance A was obtained by separating it using the above method.

[0019] This snake venom substance A has anti-tumor effects, especially inhibiting cervical cancer, nasopharyngeal carcinoma, and gastric cancer, and has relatively high safety, making it a potential drug for anti-tumor treatment, particularly for cervical cancer, nasopharyngeal carcinoma, and gastric cancer.

[0020] Compared with the prior art, the technical effects of this invention are reflected in:

[0021] (1) The method of this application can replace the existing packing material to complete the separation of snake venom and obtain cobopeptide and other snake venom substances.

[0022] (2) The method of this application can not only obtain cobo peptides, but also other effective substances of snake venom, thereby improving the utilization rate of snake venom and laying the foundation for the effective development and utilization of other substances of snake venom.

[0023] (3) The method of this application obtains snake venom substance A while obtaining cosmopeptide. Snake venom substance A has anti-tumor effects, especially the ability to inhibit cervical cancer, nasopharyngeal carcinoma and gastric cancer. It has relatively high safety and has the potential value of being developed into an anti-tumor drug, especially for cervical cancer, nasopharyngeal carcinoma and gastric cancer. Attached Figure Description

[0024] Figure 1 This is a purity spectrum of snake venom (C8).

[0025] Figure 2 This is a snake venom isolation spectrum (280nm).

[0026] Figure 3 This is the purified spectrum of Cobopeptide (280nm).

[0027] Figure 4 This is a purity spectrum of Cobopeptide (C18).

[0028] Figure 5 This is the purity spectrum of substance A (C8).

[0029] Figure 6 This refers to the effect of substance A on the proliferation of HeLa cells.

[0030] Figure 7 This refers to the effect of substance A on the proliferation of HONE1 cells.

[0031] Figure 8 This describes the effect of substance A on the proliferation of SGC7901 cells.

[0032] Figure 9 This refers to the effect of substance A on the proliferation of LO2 cells.

[0033] Figure 10 This shows the effect of substance A on HeLa cell migration. Figure A is a microscopic observation, and Figure B is a statistical graph.

[0034] Figure 11 The effect of substance A on HONE1 cell migration is shown in Figure A, which is a microscopic observation, and Figure B is a statistical graph.

[0035] Figure 12 The figure shows the effect of substance A on the migration of SGC7901 cells. Figure A is a microscopic observation and Figure B is a statistical graph.

[0036] Figure 13 The effect of substance A on HeLa cell apoptosis was detected by flow cytometry.

[0037] Figure 14 This is a statistical graph showing the proportion of HeLa cells treated with substance A in different quadrants.

[0038] Figure 15 The effect of substance A on apoptosis in HONE1 cells was detected by flow cytometry.

[0039] Figure 16 This is a statistical graph showing the proportion of HONE1 cells treated with substance A in different quadrants.

[0040] Figure 17 The effect of substance A on apoptosis in SGC7901 cells was detected by flow cytometry.

[0041] Figure 18 This is a statistical graph showing the proportion of SGC7901 cells treated with substance A in different quadrants. Detailed Implementation

[0042] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0043] Example

[0044] Preparation process for extracting cobopeptide and snake venom substance A from snake venom

[0045] Research approach:

[0046] Cobratide is a neurotoxin protein isolated and purified from the venom of the Chinese cobra. It is a polypeptide drug used for chronic pain such as late-stage cancer pain, chronic joint pain, and sciatica. Its primary structure consists of 62 amino acids with four groups of disulfide bonds, an isoelectric point of pH 8.8, and an amino acid sequence of: LECHNQQSSQ TPTTTGCSGG ETNCYKKRWRDHRGYRTERG CGCPSVKNGI EINCCTTDRC NN. Its molecular weight is 6944.9925 Da. Cobratide accounts for approximately 6% of the dried cobra venom powder from Zhoushan cobra, while approximately 94% of other substances in the venom have not yet been developed and utilized.

[0047] I. Project Research Status:

[0048] 1. Sample Information

[0049] snake venom 7.81 175

[0050] 2. Method Design / Experimental Principles

[0051] Based on the isoelectric point, molecular weight, and relative molecular mass of salts in the buffer solution of Cobropeptide, the crude extraction stage uses ion exchange packing material or other packing materials (affinity, hydrophobic, etc.) with a globulin separation range (Da) of 6944.9925 Da to separate Cobropeptide and other substances. The purification stage uses dextran or agarose packing material to desalt or concentrate Cobropeptide and other substances.

[0052] 3. Experimental instruments

[0053] LC1260 liquid chromatograph

[0054] Column: SymmetryShoeld RPC183.9*150mm, 5µm, Waters

[0055] Column: Analytical 4.6*150mm 5-Micron

[0056] Chromatography empty columns (16*400, 16*1000, 26*400, 10*600)

[0057] Plunger pumps (10ml, 50ml)

[0058] T1000 UV detector

[0059] Automatic Partial Collector BS-100A

[0060] Whatman Filter 3-Piece Set

[0061] 4. Materials and reagents

[0062] TFA (Sigma)

[0063] CAN (Fisher Chemica)

[0064] H2O (Watsons, self-produced purified water, self-produced water for injection)

[0065] NaCl (Jiangsu Qinfen)

[0066] Na2HPO4 (Chengdu Huayi)

[0067] NaH2PO4 (Chengdu Huayi)

[0068] HCl (Hunan Erkang)

[0069] 5. Preliminary experiment:

[0070] We explored various packing materials from both domestic and international sources to conduct small-scale tests on snake venom separation. We screened out packing materials that could separate coprolites with relatively high recovery rates and then optimized and studied the separation conditions.

[0071] 5.1 Packing Material Details:

[0072] 15SP-HNmicro (5ml pre-packed column)

[0073] Geldex 30PG (200ml)

[0074] Puredex LH-20 (100g)

[0075] Puredex G-50F (100g)

[0076] Puredex G-50SF (100g)

[0077] Puredex G-25F (100g)

[0078] CM C-25 (100g)

[0079] LXPM CM706M (5ml pre-filled column)

[0080] LXMS 50HQ (5ml pre-filled column)

[0081] LXMS15Q (5ml pre-filled column)

[0082] LXMS 30Q (5ml pre-filled column)

[0083] LH-20 (100g)

[0084] CM-32 (100g)

[0085] Proteomin POR15-S (5ml pre-packed column, 4.6*150mm pre-packed column)

[0086] Proteomin POR30-S (5ml pre-packed column)

[0087] Proteomin POR15-S (300ml)

[0088] 5.2 Implementation of Preliminary Experiments

[0089] 5.2.1 Preparations before separation:

[0090] 5.2.1.1 Preliminary Experiment on Snake Venom Isolation

[0091] 5.2.1.1.1 Pre-packed column: After unpacking the pre-packed column, replace the protective solution in the column with 3-5 CV of water for injection, and then perform online CIP according to the packing instructions. Only after the treatment is completed can the preliminary experiment be carried out. The buffer concentration used in the preliminary experiment is pH=7.8-8.0 phosphate buffer containing 0.01-1M sodium chloride.

[0092] 5.2.1.1.2 Dry powder or wet gel packing: PZ10*600 column is used for self-packing. Packing is carried out according to the packing instructions and manufacturer's recommendations. After packing, online CIP is performed according to the packing instructions for future use. The buffer concentration used in the preliminary experiment is pH=7.8~8.0 phosphate buffer containing 0.01-1M sodium chloride.

[0093] 5.2.1.1.3 Snake venom and cyproterone detection: The purity of the snake venom was tested before separation (C8, purity chart see...). Figure 1 The content of kobopeptide in snake venom was calculated by comparing the peak area with that of the reference standard. After separation, the purity (C18) and content of crude extract or refined liquid / powder (acetone sample, lyophilized powder) of kobopeptide were also tested. The recovery rate, desalination rate and content of the entire separation process were calculated by comparing the results before and after.

[0094] 5.2.1.1.3.1 Purity testing (C18) method:

[0095] Mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was 0.1% TFA and 50% CAN aqueous solution. The sample was loaded by an autosampler and then separated by a chromatographic column at a flow rate of 1.5 ml / min, a UV detection wavelength of 214 nm, a column temperature of 45 ℃, and a separation time of 45 min. The gradient is shown in Table 1.

[0096] Table 1. Separation Gradients

[0097] 0 92 8 4 92 8 34 60 40 38 60 40 45 92 8

[0098] For the assay, inject 50 μL of the sample into the liquid chromatograph, record the chromatogram until twice the retention time of the main component, and calculate the purity using the peak area normalization method. The purity should not be less than 90%.

[0099] 5.2.1.1.3.2 Purity Testing (C8) Method:

[0100] Mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was 0.1% TFA and 50% CAN aqueous solution. Gradient elution was performed at a flow rate of 1 ml / min, a UV detection wavelength of 214 nm, a column temperature of 36 ℃, and a separation time of 38 min. The gradient is shown in Table 2.

[0101] Table 2. Separation Gradients

[0102] 0 92 8 4 92 8 16 0 100 22 0 100 28 92 8 38 92 8

[0103] For the assay, inject 50 μL of the sample to be tested into the liquid chromatograph, record the chromatogram until the retention time of the main component is twice the value, and calculate the purity of the crude extract or finished product of Cobopeptide using the peak area normalization method. The purity should not be less than 90%.

[0104] 5.2.1.2 Preliminary Experiment of Kebopeptide Refining Section

[0105] 5.2.1.2.1 The refined section packing material collected or purchased for this project is either dry powder or wet gel packing material: XK10*600 columns are selected for self-packing. After packing, online CIP is performed according to the packing material instructions for future use. The buffer concentration used in the preliminary experiment is water for injection, and the raw material is recovered low-purity Cobropeptide. After separation, each group of components is collected for purity testing. The purity and content changes of Cobropeptide before and after sample loading are compared with those extracted by the packing material used in the existing process to see if they meet the requirements.

[0106] 5.2.2.1 Sample preparation:

[0107] 5.2.2.1.1 Snake venom treatment: Take dried snake venom powder, dissolve it with 0.01M phosphate to prepare a 0.2g / ml solution, filter it through a 0.45um membrane, and put 2ml of the filtrate into a 2ml sample vial for separation.

[0108] 5.2.2.1.2 Treatment of Cobopeptide: Take the recovered low-purity Cobopeptide dry powder, dissolve it with water for injection, prepare a 0.2 g / ml solution, filter it through a 0.45 μm membrane, take 2 ml of the filtrate and put it into a 2 ml sample vial for separation.

[0109] 5.2.2.2 Separation Operation:

[0110] 5.2.2.2.1 Snake venom isolation: Take a pre-packed or self-packed column, equilibrate it with 3CV of 0.01M phosphate buffer, 0-1M NaCl phosphate buffer, and elute with 3 column volumes. The injection volume is 0.5ml. Collect the drug solution of each component for C8 detection (elution time) to identify the target substance.

[0111] 5.2.2.2.1. Purification of Cobopeptide: Take a pre-packed or self-packed column, equilibrate it with 3CV of water for injection, process it with 1 column volume, inject 0.5 ml, flow rate 1 ml / min, collect one tube every 3 minutes, and perform C18 detection (elution time) on each tube of drug solution.

[0112] 5.2.3 Results:

[0113] 5.2.3.1 Snake venom isolation: Take the component drug solution for C8 detection and identification (peak time) to identify the target substance.

[0114]

[0115] 5.2.3.1. Purification of Cobopeptide: Collect the peak immediately upon elution, and test the purity of each tube of drug solution. Collect a larger amount of drug solution than before loading the sample.

[0116]

[0117] 5.2.4 Conclusion

[0118] 5.2.4.1. Proteomin POR15-S was used to separate Cobopeptide with a purity of approximately 96%, and the recovery rate was approximately 6% after comparing the purity before and after the separation.

[0119] 5.2.4.2, CM C-25 separation yielded a cyprotein with a purity of approximately 86.5%, and the recovery rate was approximately 8% based on the purity before and after comparison.

[0120] 5.2.4.3 LH-20 was used to separate Cobropeptide with a purity of approximately 52.01% to obtain Cobropeptide solution with a purity of 84.59%, which increased the purity by approximately 32.58% and the recovery rate was 71.4%.

[0121] 5.2.4.5 After the separation of Cobropeptide, the Proteomin POR15-S packing material can continue to elute substance A in the regeneration section.

[0122] 5.2.4.6 Design a separation route based on the results:

[0123] Coarse extraction was performed using Proteomin POR15-S. LH-20 was used for desalting and concentration, with a recovery rate of approximately 6% of the crude toxicity.

[0124] 6. Development of separation methods

[0125] 6.1 Target packing material:

[0126] 6.1.1 Target packing material for the coarse extraction section

[0127] Proteomin POR15-S

[0128] 6.1.2 Target packing material for the refining section

[0129] LH-20

[0130] 6.2 Design of extraction conditions for Cobopeptide:

[0131] 6.2.1 Determination of the pH of the initial buffer solution: Based on the isoelectric point (pI: 8.8) of Cobropeptide, the packing material (15S cation), and the Cobropeptide production process, a 0.01M phosphate buffer solution (hereinafter referred to as 0.01M buffer) with pH = 7.8–8.0 was selected as the initial buffer solution. 1530 ml of source packing material was packed into a 10*600 mm column. The packing material was prepared according to the packing material instructions (0.5M). Clean the column with NaOH solution (2-3 CV), remove the packing material from the column and transfer it to a three-piece set (use 5µm Carbide filter paper). Replace the filter paper with 0.01M buffer 3-5 times, drain the filtrate, and soak the packing material in a beaker with 0.01M buffer for 30 minutes. Take five 15*150 test tubes and place them on a test tube rack, labeling them No. 1, No. 2, No. 3, No. 4, and No. 5. Add 1ml of packing material (wet gel state) to each of the five test tubes, with different pH values ​​(No. 1 pH = 5.5, No. 2 pH = 6.0). 2 ml of 0.01 M buffer solution (pH=6.5 for tube 3, pH=7.0 for tube 4, and pH=7.8 for tube 5) was added and shaken to suspend the sample. Then, 1 ml of Cobropeptide (prepared from Cobropeptide (sample 1) to a 30 mg / ml solution) solution was added and shaken to suspend the sample again. The sample was placed in a medical refrigerator and allowed to stand for 90 min. The supernatant was then collected for OD detection (comparing the OD values; the smaller the OD value, the more Cobropeptide was adsorbed by the packing material at that pH). Tube 5 had the smallest OD. The experiment was repeated 3 times to determine pH=7.8 as the initial pH.

[0132]

[0133] 6.2.2 Determination of Initial Buffer Salt Concentration: Take 5 15*150 test tubes and place them on a test tube rack, labeling them as No. 1, No. 2, No. 3, No. 4 and No. 5. Add 1 ml of filler (state: wet gel) to each of the 5 test tubes, then add 2 ml of 0.01M buffer solution with different salt (NaCl (Jiangsu Qinfen)) concentrations (No. 1 0M, No. 2 0.01M, No. 3 0.02M, No. 4 0.03M, No. 5 0.04M), shake well to suspend, then add 1 ml of Cobropeptide (prepared from Cobropeptide (sample 1) into a 30 mg / ml solution), shake well again to suspend, and place in a medical refrigerator for 90 min. Take the supernatant for OD detection (compare the OD values; the smaller the OD value, the more Cobropeptide is adsorbed by the filler at that salt concentration). Tube No. 1 has the smallest OD. Repeat 3 times to determine 0M as the initial buffer concentration.

[0134]

[0135] 6.2.3 Determination of Elution Salt Concentration: Take five 15*150 test tubes and place them on a test tube rack, labeling them as No. 1, No. 2, No. 3, No. 4, and No. 5. Add 1 ml of filler (state: wet gel) to each of the five test tubes, 1 ml of Cobopeptide (prepared from Cobopeptide (sample 1) to a 30 mg / ml solution), and 2 ml of 0.01 M buffer solution. Shake well to suspend the contents. Add 3 ml of 0.01 M buffer solution with different concentrations of NaCl (Jiangsu Qinfen) (No. 1: 0.02 M, No. 2: 0.04 M, No. 3: 0.06 M, No. 4: 0.08 M, No. 5: 0.1 M). Shake well again to suspend the contents. Place the tubes in a medical refrigerator and let them stand for 90 min. Take the supernatant and perform OD detection (compare the OD values; the larger the OD value, the better the desorption / elution effect at that salt concentration). The OD value of tube No. 5 is the largest. Repeat the test three times to determine 0.1 M as the elution salt concentration.

[0136]

[0137] 6.2.4 Flow Rate Determination: Pack 100ml of packing material into a column (XK16*400m). After packing, perform online CIP according to the packing material instructions. Equilibrate with 0.01M buffer solution for 5 CV. Set the instrument alarm pressure to 4 bar (column pressure resistance 4 bar). Test the flow rate at 1ml / min, 2ml / min, 3ml / min, and 4ml / min. For each condition, 1 CV was applied. The device alarmed and stopped the test. At 4ml / min, the device alarmed and stopped after 5 minutes and 34 seconds. The pressure was kept constant at 3.4 bar ± 0.1 bar at 3ml / min. Referring to the packing material instructions, the Proteomin POR15-S packing material has a pressure resistance of 100 bar. Within the column pressure resistance range, 3ml / min was selected as the process flow rate.

[0138] 6.2.5 Regeneration (Cleaning): Take eight 15*150 test tubes and place them on a test tube rack, labeling them No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, and No. 8. Add 1 ml of filler (state: wet gel), 1 ml of snake venom solution (dissolved in 0.01 M phosphate to prepare a 0.2 g / ml solution), and 2 ml of 0.01 M buffer to each of the eight test tubes. Shake well to suspend the venom. Add salts (NaCl (Jiangsu Qinfen)) with different concentrations (No. 1: 0.05 M, No. 2: 0.10 M, No. 3: 0.15 M, No. 4: 0.20 M, No. 5: 0.25 M, No. 6: 0.50 M, No. 7: 0.75 M, No. 8: 1 M) to each test tube. Add 3 ml of 0.01 M buffer solution, shake well to resuspend, and place in a medical refrigerator for 90 min. Take the supernatant for OD detection (compare the OD values; the larger the OD value, the better the desorption / elution effect at that salt concentration). Repeat 3 times. The OD values ​​of tubes 5, 6, 7, and 8 are similar to those of the blank (0.2 g / ml snake venom solution), indicating that the elution salt concentration of 0.5 M can fully elute the snake venom adsorbed on the packing material and separate substance A. Based on the packing material regeneration method in the instructions (select 1-2 M NaCl for regeneration), the elution salt concentration of 1 M NaCl solution is selected as the regeneration elution endpoint.

[0139]

[0140] 6.2.6 Gradient Optimization: Pack 100ml of packing material into a column (XK16*400m). After packing, perform online CIP according to the packing material instructions. Equilibrate with 0.01M buffer at 5CV. Elute at a gradient of 0.01M-0.05M-0.1M-1M, 5CV, 3ml / min. Elute at an isogradient of 0.01M→0.05M→0.1M→1M, 1CV / isogradient, 3ml / min. Repeat 3 times. The 0.01M-0.1M gradient has a shorter separation time, higher recovery rate, and can separate more effective substances from snake venom (13 peaks under the 0.01M-0.1M-1M gradient condition, and 8 peaks under the 0.01M→0.05M→0.1M→1M isogradient condition).

[0141] 6.2.7 Methodological Validation:

[0142] 6.2.8.1 Reproducibility: In three consecutive repeated experiments, Cobopeptide showed a peak at 72±2 min, and substance A showed a peak at 98±1 min.

[0143] 6.2.9.2 Stability: The column pressure and packing height were statistically analyzed in three consecutive repeated tests. The column pressure was maintained at 3.5±0.2 bar, and the packing height remained unchanged after regeneration.

[0144] 6.2.7 Experiment Implementation

[0145] 6.2.7.1 Take dried snake venom powder, dissolve it with 0.01M phosphate to prepare a 0.5g / ml solution, filter it through a 0.45um membrane, and inject 5ml of the filtrate into a 5ml sample loop for separation.

[0146] 6.2.7.2 Final Chromatographic Conditions: Separation conditions: Mobile phase A was 0.01M phosphate buffer, and mobile phase B was phosphate buffer containing 1M NaCl. Samples were automatically extracted by the equipment. Injection volume was set to 5 ml, flow rate to 3 ml / min, UV detection wavelength to 280 nm, and separation time to 125 min. The gradient is shown below, and the separation chromatogram is shown in the figure. Figure 2 .

[0147] 0 100 0 25 95 5 75 90 10 100 0 100 101 100 0 125 100 0

[0148] 6.2.7.3 After separation, collect peaks according to the spectrum (Note: for cross peaks, discard the sample 1 min before and after the peak bottom), combine the drug solutions, measure the volume, and set aside for use.

[0149] 6.2.7.4 Treatment of the chemical solution

[0150] Add 3 times the volume of cold acetone to the previous solution, place it in a refrigerator for 30 minutes for cold precipitation, remove it and filter it. After filtration, place the filter cake in a freeze dryer for low-temperature drying.

[0151] 6.2.7.5 Drug Collection

[0152] Once the vacuum degree of the freeze dryer is less than 1 Pa and the freeze-drying time is greater than 24 hours, remove the dried cake, grind it, and weigh it.

[0153] 6.3 Refining process conditions design: The main purpose of the refining section is concentration and desalting. Salt-free solution (water for injection) is selected as the eluent. The crude extract is packed into the column and concentrated and desalted according to the flow rate recommended in the packing instructions.

[0154] 6.3.1 Separation conditions: For purification operations, select an XK16*1000 column (adjustable at both ends), and load... LH-20 packing material, column height controlled at 70 cm. Before sample loading, the column was cleaned with 0.5 M hydroxide solution using 1 CV, followed by equilibration with 5 CV of water for injection. Mobile phase A was water for injection. Injection volume was set to 5 ml, flow rate to 2 ml / min, UV detection wavelength to 280 nm, separation time to 70 min. The gradient is shown below, and the separation chromatogram is shown in the figure. Figure 3 .

[0155] 0 100 70 100

[0156] 6.3.2 Collection of medicinal solution

[0157] After separation, peaks were collected according to the spectrum (Note: for cross-peaks, samples 1 min before and after the peak bottom were discarded), the drug solutions were combined, the volume was measured, and the solution was ready for use.

[0158] 6.3.3 Treatment of the chemical solution

[0159] Pour the above-mentioned liquid medicine into a freeze-drying pan, place it in a cold trap for pre-freezing, and wait until the liquid medicine is completely solidified and the sample temperature is below -50℃. Then turn on the vacuum to dry the sample. When the vacuum degree of the freeze dryer is less than 1pa and the freeze-drying time is greater than 24h, take out the dried cake, grind it, weigh it and take samples for testing.

[0160] 6.4 Results:

[0161] 6.4.1. The purity of Kebo peptide is shown in [reference needed]. Figure 4 ;

[0162] 6.4.2. The purity of snake venom substance A is shown in [reference needed]. Figure 5 .

[0163] Experimental study on the in vitro antitumor effect of substance A:

[0164] 1. Materials

[0165] 1.1 Test Sample

[0166] Substance A is the snake venom substance A extracted earlier.

[0167] 1.2 Cell lines

[0168] (1) HeLa cells, a human cervical cancer cell line, were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

[0169] (2) Human nasopharyngeal carcinoma cell line HONE1 was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (3) Human gastric carcinoma cell line SGC7901 was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (4) Human normal hepatocyte cell line LO2 was preserved in the laboratory.

[0170] 1.3 Main Instruments and Consumables

[0171] (1) CO2 constant temperature incubator: Thermo Corporation, USA, Model 310.

[0172] (2) Inverted microscope: Leica, Germany, model DM 1L LED.

[0173] (3) Clean bench: Suzhou Purification Equipment Co., Ltd., model SW-CJ-2D.

[0174] (4) Centrifuge: Shanghai Anting Scientific Instrument Factory, model TGL-16G.

[0175] (4) Multifunctional microplate reader: Biotech Synergy, USA.

[0176] (5) Flow cytometer: Agilent Technologies, NovoCyte.

[0177] (6) Horizontal shaking table: Qilinbei Instrument Manufacturing Co., Ltd., TS-8 model.

[0178] (7) Cell culture flasks: Corning, Inc., USA.

[0179] (8) 96-well plate: Wuxi Nice Life Science & Technology Co., Ltd.

[0180] 1.4 Main Reagents

[0181] (1) DMEM (high sugar) medium: HyClone.

[0182] (2) Fetal bovine serum (FBS): Gibico.

[0183] (3) Pancreatic enzyme: Ameresco, USA.

[0184] (4) Cell Counting Kit-8 (CCK8) reagent kit: APExBIO Inc., USA.

[0185] (5) Apoptosis kit: Nanjing Kaiji Biotechnology Development Co., Ltd.

[0186] 2. Methods

[0187] 2.1 Cell Culture

[0188] Frozen HeLa, HONE1, SGC7901, and LO2 cells were rapidly thawed in a 37°C water bath. The cryovials were cleaned with cotton balls soaked in 75% alcohol, transferred to a clean bench, and the cell suspension was slowly aspirated into 5mL centrifuge tubes. 1mL of complete culture medium (DMEM containing 10% fetal bovine serum and 1% antibiotics) was added, and the tubes were centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded, and the cells were collected. The cells were resuspended in 1mL of complete culture medium and evenly seeded into culture flasks containing 5mL of preheated 37°C complete culture medium. The flasks were gently shaken to ensure even distribution of cells. The cells were observed under an inverted microscope. The flasks were then incubated at 37°C with 5% CO2, and the medium was changed every 24 hours.

[0189] When cells reach 80%–90% confluence, they are passaged. For passage, first aspirate the old culture medium, wash twice with 1–2 mL PBS, add 0.5 mL of 0.25% trypsin, digest at 37°C for 3 min, centrifuge at 1000 rpm for 3 min, aspirate the supernatant, add 1 mL of complete culture medium, and gently pipette to disperse the cells evenly. Take 0.5 mL of this mixture and add it to two culture flasks containing 5 mL of preheated 37°C complete culture medium (DMEM medium containing 10% fetal bovine serum and 1% antibiotics). Gently shake to distribute the cells evenly in the flasks, and incubate at 37°C with 5% CO2. Passage every 2–3 days until the cells reach the logarithmic growth phase, then use them for experiments.

[0190] 2.2 CCK8 Experiment

[0191] HeLa cells and HONE1 / SGC7901 cells in logarithmic growth phase were collected, digested with 0.25% trypsin, and the cells were prepared into a cell suspension. 100 μL / well was seeded into 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The culture medium was discarded, and the cells were washed twice with PBS. Culture medium containing substance A at concentrations of 800 μg / mL, 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, and 3.125 μg / mL were added, with six replicates for each concentration. Six replicates of the control well (without the drug) and a zeroing well containing the drug but without cell seeding were also included. After 24 hours of drug treatment, 10 μL of CCK8 solution was added to each well, and the cells were incubated for 2 hours. The absorbance (OD) value at 450 nm was measured using a microplate reader, and the cell proliferation rate after treatment with different concentrations of substance A was calculated using the following formula. The experiment was repeated three times.

[0192]

[0193] Logarithmic growth phase LO2 cells were seeded into 96-well plates as described above. The concentrations of substance A added were 6400 μg / mL, 3200 μg / mL, 1600 μg / mL, 800 μg / mL, 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, and 25 μg / mL, respectively. The remaining procedures were the same as above.

[0194] The obtained data were input into Prism 7 software to calculate the IC50 of substance A on 3 tumor cell lines and 1 normal cell line. 50 value.

[0195] 2.3 Transwell cell migration assay

[0196] HeLa cells, HONE1 cells, and SGC7901 cells in logarithmic growth phase were collected, digested with 0.25% trypsin, and resuspended in serum-free culture medium to prepare cell suspensions. The density of HeLa cells was adjusted to 5 × 10⁶ cells / year. 5 The density of HONE1 cells / mL and SGC7901 cells was adjusted to 1×10⁻⁶. 5 Cells / mL. 200 μL of cell suspension from each of the three cell lines was added to the upper chamber of the Transwell chamber. 600 μL of drug-containing culture medium (containing 10% fetal bovine serum) with different concentrations of substance A was added to the lower chamber. The concentrations of substance A added to the lower chamber for HeLa cells were 62.5, 125, and 250 μg / mL; for HONE1 cells, the concentrations were 4.5, 9, and 18 μg / mL; and for SGC7901 cells, the concentrations were 9.5, 19, and 38 μg / mL. Three replicates were set for each concentration. Cells were cultured for 24 h. The Transwell chamber was then removed, washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, stained with 0.1% crystal violet, and the cells in the upper chamber were wiped off. Cells migrating to the lower chamber were observed under a microscope. Five fields of view (center and perimeter) were counted, and images were taken under an inverted microscope. Cell counts were recorded using ImageJ software, and the average value was taken.

[0197] 2.4 Flow Cytometry Detection of Apoptosis

[0198] HeLa cells, HONE1 cells, and SGC7901 cells in logarithmic growth phase were collected after trypsin digestion and prepared to a cell density of 1×10⁻⁶ cells / cells. 5Cell suspension at 2 mL / well was added to 6-well plates. When the cell density reached approximately 80%, each type of tumor cell was divided into a control group and low-, medium-, and high-dose groups, with three replicates per group. Low, medium, and high doses of substance A were then added to each group to treat the cells. The low, medium, and high doses of substance A added to HeLa and HONE1 cells were 25, 50, and 100 μg / mL, respectively; the low, medium, and high doses of substance A added to SGC7901 cells were 0, 37.5, 75, and 150 μg / mL, respectively. The control group received culture medium without substance A. After 24 hours of cell culture following drug addition, the cell culture medium was collected into 2 mL centrifuge tubes. Wash cells twice with PBS, digest cells with trypsin, stop trypsin digestion with the collected cell culture medium, gently pipette the cells, transfer them to centrifuge tubes, and centrifuge at 1000 rpm for 5 min; discard the supernatant, collect the cell pellet, wash cells twice with PBS, and centrifuge at 1000 rpm for 5 min; discard the supernatant, gently resuspend the cells in 500 μL Binding Buffer; add 5 μL Annexin V-FITC and mix slowly; then add 5 μL propidium iodide staining solution and mix slowly; incubate in the dark at room temperature for 5-10 min, and then analyze using flow cytometry.

[0199] 3. Results

[0200] 3.1 CCK8 Experimental Results

[0201] Substance A inhibits the IC50 of various cell proliferations. 50 The values ​​and their 95% confidence periods are shown in Table 3. The effect of substance A on the proliferation of each cell is shown in Table 3. Figures 6-9 As shown in the figure. The results showed that substance A significantly inhibited the proliferation of three types of tumor cells: HeLa cells, HONE1 cells, and SGC7901 cells. Substance A had a relatively weak inhibitory effect on the proliferation of normal human hepatocytes (LO2 cells). The IC50 value of substance A in inhibiting LO2 cell proliferation was [not specified]. 50 The value is much greater than the IC50 value of substance A in inhibiting tumor cell proliferation.

[0202] Table 3 shows the IC50 values ​​of substance A in inhibiting the proliferation of various cells. 50 Value and 95% confidence limit

[0203]

[0204] 3.2 Results of Transwell cell migration assay

[0205] 3.2.1 Results of the effect of substance A on HeLa cell migration

[0206] The effect of substance A on HeLa cell migration is shown in [reference needed]. Figure 10 A and Figure 10B. The results showed that substance A significantly inhibited HeLa cell migration, and the inhibitory effect increased with increasing drug concentration.

[0207] 3.2.2 Results of the effect of substance A on HONE1 cell migration

[0208] The effect of substance A on HONE1 cell migration is shown in [reference needed]. Figure 11 A and Figure 11 B. The results showed that substance A significantly inhibited HONE1 cell migration, and the inhibitory effect increased with increasing drug concentration.

[0209] 3.2.3 Effects of Substance A on SGC7901 Cell Migration

[0210] The effect of substance A on the migration of SGC9701 cells is shown in the figure. Figure 12 A and Figure 12 B. The results showed that substance A significantly inhibited the migration of SGC7901 cells, and the inhibitory effect increased with increasing drug concentration.

[0211] 3.3 Flow cytometry results

[0212] 3.3.1 Effect of substance A on HeLa cell apoptosis

[0213] HeLa cells treated with substance A were analyzed by flow cytometry. The results showed that in the control cells without substance A, normal cells (distributed in the lower left quadrant) predominated, accounting for an average of 95.38%, while the proportions of early apoptotic cells (distributed in the lower right quadrant), late apoptotic cells (distributed in the upper right quadrant), and necrotic cells (distributed in the upper left quadrant) were very small. After treatment with different doses of substance A, the proportion of normal cells decreased with increasing substance A dose, while the proportions of early apoptotic cells, late apoptotic cells, and necrotic cells all increased with increasing substance A dose, with the increase in the proportion of necrotic cells being more pronounced. Among the treatments, the proportion of normal cells in HeLa cells treated with low, medium, and high doses of substance A was significantly lower than that in the control cells, with statistically significant differences (P < 0.01). Only the proportion of early apoptotic cells in HeLa cells treated with high doses of substance A was significantly increased compared to the control, with a statistically significant difference (P < 0.01). The proportions of late apoptotic and necrotic cells in HeLa cells treated with low, medium, and high doses of substance A were significantly increased compared to the control cells, with statistically significant differences (P < 0.05 or P < 0.01). Results are shown below. Figure 13 Table 4 Figure 14 .

[0214] Table 4. Proportion of HeLa cells in different quadrants after treatment with substance A.

[0215]

[0216] 3.3.2 Effect of substance A on apoptosis in HONE1 cells

[0217] After treating HONE1 cells with substance A, flow cytometry analysis showed that the control cells without substance A treatment were predominantly normal cells, accounting for an average of 92.36%, with very small proportions of early apoptotic, late apoptotic, and necrotic cells. After treatment with substance A, the proportion of normal cells decreased with increasing substance A dosage, while the proportions of early apoptotic, late apoptotic, and necrotic cells all increased with increasing substance A dosage, with the increase in necrotic cells being more pronounced. Among the treatments, the proportion of normal cells in HONE1 cells treated with low, medium, and high doses of substance A was significantly lower than that in the control cells, with statistically significant differences (P < 0.05 or P < 0.01). The proportion of early apoptotic cells in HONE1 cells treated with any of the three doses of substance A was not significantly different from that in the control cells. The proportion of late apoptotic cells in HONE1 cells treated with medium and high doses of substance A was significantly increased compared to the control cells, with statistically significant differences (P < 0.05), but the proportion of late apoptotic cells in HONE1 cells treated with low doses of substance A was not significantly different from that in the control cells. The proportion of necrotic cells in HONE1 cells treated with low, medium, and high doses of substance A was significantly increased compared to the control cells, with statistically significant differences (P < 0.05 or P < 0.01). Results are shown below. Figure 15 Table 5 Figure 16 .

[0218] Table 5. Proportion of HONE1 cells in different quadrants after treatment with substance A.

[0219]

[0220] 3.3.3 Effect of substance A on apoptosis in SGC7901 cells

[0221] After treating SGC7901 cells with substance A, flow cytometry analysis showed that the control cells without substance A treatment were mainly normal cells, with an average proportion of 92.46%, and the proportions of early apoptotic, late apoptotic, and necrotic cells were very small. After treating SGC7901 cells with substance A, the proportion of normal cells decreased with increasing substance A dosage, the proportion of early apoptotic cells did not change significantly, while the proportions of late apoptotic and necrotic cells increased with increasing substance A dosage, with the increase in the proportion of necrotic cells being more significant. Specifically, after treating SGC7901 cells with low, medium, and high doses of substance A, the proportion of normal cells was significantly lower than that of control cells, and the differences were statistically significant (P < 0.01); after treating SGC7901 cells with low, medium, and high doses of substance A, the proportions of late apoptotic and necrotic cells were significantly increased compared with control cells, and the differences were statistically significant (P < 0.05 or P < 0.01). Results are shown below. Figure 17Table 6 Figure 18 .

[0222] Table 6. Proportion of SGC7901 cells in different quadrants after treatment with substance A.

[0223]

[0224]

[0225] 4. Discussion

[0226] To investigate whether substance A has anti-tumor effects, this experiment examined the effects of substance A on the proliferation and migration of three tumor cell lines: HeLa cells, HONE1 cells, and SGC7901 cells, in vitro. The results showed that substance A significantly inhibited the proliferation of all three tumor cell lines. 50 The values ​​were 120.4 μg / mL, 9.07 μg / mL, and 18.22 μg / mL, respectively.

[0227] If a drug significantly inhibits both tumor cell proliferation and normal cell proliferation, it indicates a lack of selectivity in its inhibitory effect on tumor cells, potentially suggesting low safety. Conversely, if a drug inhibits tumor cell proliferation while having minimal impact on normal cells, it indicates high safety and greater potential for development and application. Therefore, this study used the human normal hepatocyte cell line LO2 as the subject to observe the effect of substance A on normal cell proliferation. The results showed that substance A required a relatively high concentration to inhibit LO2 cell proliferation, and the IC50 of substance A in inhibiting LO2 cell proliferation was [not specified in the original text]. 50 The value was 1029 μg / mL, which is much higher than the IC50 value for inhibiting tumor cell proliferation. 50 The value indicates that substance A has a significantly stronger effect on inhibiting the proliferation of tumor cells than normal cells, suggesting that substance A has a certain selectivity for tumor cells and good safety.

[0228] Based on the observation that substance A has a significant inhibitory effect on HeLa cells, HONE1 cells, and SGC7901 cells, this study further investigated the effect of substance A on the migration ability of these three cell lines. The results showed that, compared with the control group without drug administration, the migration ability of HeLa cells, HONE1 cells, and SGC7901 cells was significantly weakened after the addition of different concentrations of substance A. Furthermore, the weakening of migration ability became more pronounced with increasing substance A dosage, exhibiting a certain dose-dependent effect.

[0229] This study investigated the effects of substance A on apoptosis in HeLa, HONE1, and SGC7901 cells. Annexin V / PI double staining and flow cytometry were used to detect apoptosis in these three tumor cell types after treatment with substance A. The Annexin V / PI double staining method can detect the proportions of normal cells, early apoptotic cells, late apoptotic cells, and necrotic cells. The results showed that after treatment with substance A, the proportion of normal cells decreased with increasing drug dosage, while the proportions of late apoptotic and necrotic cells increased with increasing drug dosage, with the increase in necrotic cells being more significant. This indicates that substance A can promote tumor cell apoptosis, but its tumor-killing effect is more prominent. Furthermore, the results showed that the proportion of early apoptotic cells did not change significantly after treatment with substance A. Only when HeLa cells were treated with high doses of substance A did the proportion of early apoptotic cells significantly increase compared to control cells, suggesting that substance A rapidly induces tumor cell apoptosis, quickly leading to late apoptosis after drug treatment.

[0230] 4. Summary

[0231] In summary, substance A significantly inhibited the proliferation and migration of three tumor cell lines (HeLa, HONE1, and SGC7901) and promoted apoptosis in all three cell lines, demonstrating a significant tumor-killing effect. Meanwhile, substance A had little effect on the normal human hepatocyte cell line LO2, inhibiting the IC50 of LO2 cells. 50 The value is much greater than the IC50 value for inhibiting tumor cells. 50 Value (IC value even if the sample is retested after a period of storage) 50 The value was also much smaller than the IC50 value for inhibiting LO2 cells. 50 The value indicates that substance A has relatively high safety. This study suggests that substance A has the potential value for development into an anti-tumor drug.

[0232] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A preparation process for extracting cobopeptide and snake venom substance A from snake venom, characterized in that, Snake venom was dissolved in 0.01M phosphate and eluted using ion exchange chromatography with Proteomin POR15-S packing material. The mobile phase was 0.01M phosphate buffer (A) and 1M NaCl-containing phosphate buffer (B). The flow rate was 1-5 ml / min and the detection wavelength was 280 nm. The elution is a gradient elution, and the elution gradient is as follows: Collect the eluent for 72 ± 2 minutes to obtain Cobopeptide, and collect the eluent for 98 ± 1 minute to obtain Snake Extract A.

2. The preparation process for extracting cobopeptide and snake venom substance A from snake venom as described in claim 1, characterized in that, Includes the following steps: (1) Sample preparation: Take dried snake venom powder, dissolve it with 0.01M phosphate to prepare a 0.1-1g / ml solution, filter it, and take the filtrate for separation; (2) Separation conditions: Ion exchange chromatography was used, with Proteomin POR15-S as the packing material, 0.01M phosphate buffer as mobile phase A and 1M NaCl phosphate buffer as mobile phase B for elution. (3) Separation and collection: Automatic collectors are used for collection, every 1 minute per sample. After separation, peaks are collected according to the spectrum. (4) Add acetone to the separated and collected liquid, let it settle, filter and dry to obtain the final product.

3. The preparation process for extracting cobopeptide and snake venom substance A from snake venom as described in claim 2, characterized in that, The sample was prepared by dissolving dried snake venom powder in 0.01M phosphate to prepare a 0.5g / ml solution; the flow rate was 3 ml / min.

4. The preparation process for extracting cobopeptide and snake venom substance A from snake venom as described in claim 2, characterized in that, The acetone is three times the volume of the drug solution in cold acetone; the cold precipitation is cold precipitation for 30 minutes; the filtration is vacuum filtration; and the drying is low-temperature drying.

5. The preparation process for extracting cobopeptide and snake venom substance A from snake venom as described in claim 4, characterized in that, The low-temperature drying refers to freeze drying, where the freeze dryer vacuum degree is less than 1 Pa and the freeze drying time is greater than 24 hours before removal.

6. The preparation process for extracting cobopeptide and snake venom substance A from snake venom as described in claim 2, characterized in that, In step (3), when cross peaks occur during the collection process, the samples before and after the peak bottom are discarded within 1 minute.

7. The preparation process for extracting cobopeptide and snake venom substance A from snake venom as described in claim 1, characterized in that, It also includes a purification process: Take the crudely extracted cobopeptide or snake venom substance A, dissolve it in water for injection to prepare a 0.1-1 g / ml solution, use Selife® LH-20 packing material, use water for injection as the elution solvent, elute at a flow rate of 2 ml / min, use a detection wavelength of 280 nm, elute for 70 min, collect using an automatic collector, every 1 min per vial, collect the peaks according to the spectrum after separation, and freeze dry to obtain the final product.

8. Snake venom substance A obtained by the preparation process of claim 1.

9. The use of the snake venom substance A according to claim 8 in the preparation of drugs for cervical cancer, nasopharyngeal carcinoma and gastric cancer.

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