Method for separating anti-tumor active polypeptide from Buthus martensii Karsch and application

Abstract

The invention discloses a method for separating anti-tumor active polypeptide from Buthus martensii Karsch and application thereof. The method comprises the following steps: firstly, crushing dry buthus martensii into powder, and fully degreasing with petroleum ether; extracting the degreased powder in a low-temperature dark environment, centrifuging to take a supernatant, freeze-drying to obtain a crude extract, and separating and purifying the crude extract by utilizing ion exchange chromatography and gel chromatography to obtain four buthus martensii polypeptide components which are respectively named as S4-F1, S4-F2, S4-F3 and S4-F4; and the anti-tumor activity of the four polypeptide components is detected. The result shows that the IC50 of the S4-F1 part on the HCT-116 colon cancer cell inhibition activity is 182 [mu] g / mL + / -2.97; the component S4-F1 mainly contains the molecular weight range of 6.5 kDa to 10.6 kDa, the total protein content is 81.34%, and the component S4-F1 has high anti-tumor activity. The method has the advantages that the extraction cost is reduced, the safety is improved, the extraction rate is high, the property is stable, the purity is high, the extraction time is short, and the repeatability is high. The compound can be used in the fields of antitumor drugs and health-care products.

Description

Technical Field

[0001] This invention relates to bioactive peptides, specifically to a method and application for isolating antitumor active peptides from the East Asian scorpion. Background Technology

[0002] East Asian scorpion ( Buthus martensii Scorpions (BMK) have a long history of use in food and medicine. Traditionally, they are used in medicine primarily in the form of whole scorpion powder or soaked in alcohol. Some ancient texts also record soaking whole scorpions in vegetable oil and then sun-drying them for a period of time before applying them topically. According to the *Compendium of Materia Medica*, the *Dictionary of Traditional Chinese Medicine*, and the *Chinese Pharmacopoeia*, whole scorpions have the functions of "extinguishing wind and relieving spasms, reducing inflammation and attacking toxins, and clearing the meridians and relieving pain"; they are mainly used to treat "infantile convulsions, spasms, skin diseases, cardiovascular and cerebrovascular diseases, inflammation, hepatitis B, and tumors." Records also indicate that whole scorpions have the effects of dispelling dampness and wind, strengthening muscles and tendons, softening and dissolving hardened stones, promoting diuresis and expelling stones, and opening the ear orifices. They are mainly used to treat paralysis, facial paralysis, joint pain, weakness of hands and feet, kidney stones, bladder stones, tumors, and leukoderma. Whole scorpions are also a high-end delicacy, rich in nutrients, and have functions such as disease prevention and treatment, enhancing immunity, and anti-aging. They are also a low-fat, high-protein food with excellent nutritional and health benefits, making them highly valuable for consumption. Nowadays, whole scorpions have received widespread attention as a high-value new resource food with specific health benefits.

[0003] Malignant tumors are the leading cause of death among humans, posing a significant threat to human life and health. While cancer can be alleviated and treated through surgery, chemotherapy, and radiotherapy, surgical cure rates are low, and radiotherapy and chemotherapy often cause severe side effects, frequently damaging normal cells. Therefore, the continuous search for highly effective, low-toxicity, and highly specific anti-tumor drugs is a hot topic in the medical field. As of 2022, the leading cause of cancer death in my country was lung cancer (733,300 deaths), followed by liver cancer (316,500 deaths), stomach cancer (260,400 deaths), colorectal cancer (240,000 deaths), and esophageal cancer (187,500 deaths), accounting for 67.80% of all cancer deaths. Due to their strong targeting and low cytotoxicity, protein drugs are highly suitable as precursor compounds for finding anti-tumor drugs. Currently, commonly used anti-tumor drugs mainly fall into eight categories: cytotoxic drugs, small molecule targeted drugs, antibody drugs, immune checkpoint inhibitors, hormones, antibody-drug conjugates, and others. The mechanisms by which protein drugs produce antitumor activity mainly include inhibiting cancer cell DNA replication, transcription and protein synthesis, affecting tumor cell biochemical metabolism and intracellular signal transduction pathways, affecting the expression of related oncogenes, causing disorders in the mechanisms regulating cell growth and differentiation, and regulating immune function.

[0004] A search for relevant patents using the keywords "scorpion polypeptides, scorpion proteins, and scorpion venom" yielded a total of 35 invention patents and utility model patents. Among these, 17 patent applications related to scorpion polypeptides have been granted (7 authorized), 12 patent applications related to scorpion proteins have been granted (1 authorized), and 6 patent applications related to scorpion venom have been granted (4 authorized, including 2 utility model patents). Most of these patents focus on the processes and methods for extracting and separating polypeptide and protein components from scorpion compound preparations, as well as the related activities of these compound preparations. Their activities mainly include anti-tumor, anti-pneumonia, anti-arthritis, and thrombolytic effects. Some patents also focus on the identification and application of characteristic polypeptide components from scorpions. Among these patents, application number 202410891681.9 describes the isolation of polypeptide components with anti-vitiligo activity from the East Asian scorpion (whole scorpion). This patent is based on previous research by our group and shares many similarities with this application, such as raw materials, sample pretreatment, separation and purification methods, and some evaluation indicators. However, the difference lies in the fact that the two polypeptide components with different biological activities originate from two protein components with different solubility properties. The peptides with anti-vitiligo activity are derived from the protein fraction obtained by precipitating the total crude extract solution in a 50% ammonium sulfate solution system, while the anti-tumor colon cancer active peptide fraction is derived from the fraction obtained by precipitating in an 80% ammonium sulfate solution system. Samples with different physical properties exhibit different biological activities. Application number CN202210203340.9 describes the isolation of hyaluronidase from wild-caught scorpion venom. Hyaluronidase enhances the permeability of fluids in tissues and plays an important role in controlling cancer cell invasion, inhibiting angiogenesis, and promoting wound healing. Application number CN202111489552.X describes the isolation of toxic peptides from scorpion venom and the confirmation of their biological activity. The toxic peptides obtained from scorpion venom have a molecular weight range of 3.2 kDa-7.3 kDa and exhibit strong anti-vitiligo activity. These three patents are the results of our research group's previous studies on whole scorpions and scorpion venom. Application No. 202111308160.9 is a highly active peptide segment screened from whole scorpion polypeptide extract that has a high inhibitory effect on liver cancer, osteosarcoma, etc., and can inhibit the growth of tumor cells through multiple mechanisms; Application No. 202010202660.3 is a drug developed by extracting natural polypeptides from whole scorpion and other animal and plant medicinal materials to treat or prevent diseases such as cerebral ischemia, antiplatelet aggregation, cerebral palsy, and stroke.Application No. 202010012082.7 describes an invention that uses several medicinal materials, including scorpion, centipede, leech, *Smilax china*, and silkworm, to extract polypeptide components through traditional Chinese medicine principles and ancient formulas combined with modern scientific technology to develop a product that has significant preventive effects against rheumatism, cardiovascular and cerebrovascular diseases, and cancer. Application No. 202311330640.4 describes an invention in the field of biotechnology, specifically involving a characteristic polypeptide for identifying the East Asian scorpion and its application. This invention utilizes the relevant characteristic polypeptide to identify related drugs, enabling quality control of scorpion medicinal materials, processed medicinal slices, and drugs containing the East Asian scorpion. In addition, a search using the keywords "scorpion and anti-tumor" yielded 103 related patent applications. Of these, 101 were patent applications for compound Chinese medicine preparations containing scorpion raw materials, protecting the prescription composition and efficacy. No patents used scorpion as a single research material to study anti-tumor activity.

[0005] Currently, scorpion products on the market are mainly used for anti-inflammatory, detoxifying, and pain-relieving purposes, with almost no products containing whole scorpion anti-tumor functions. Therefore, developing a low-cost, high-activity whole scorpion product has broad market economic benefits. This invention uses conventional extraction and separation methods to separate and purify peptides with anti-cancer activity, guided by in vitro anti-tumor activity tests. Currently, the development of bioactive peptides has become a hot research area both domestically and internationally. The high activity, rapid absorption, safety, and health benefits of peptides have made them popular among product developers, and the equipment and production conditions required for peptide development are simple and feasible. Therefore, the preparation of whole scorpion anti-tumor active peptides has high feasibility and broad market economic potential.

[0006] This invention demonstrates that the East Asian scorpion contains abundant proteins and polypeptides. Extracting East Asian scorpion polypeptides and combining them with its numerous biological activities to develop whole scorpion polypeptide drugs with anti-tumor functions is of great practical significance. Summary of the Invention

[0007] The purpose of this invention is to provide a method and application for isolating antitumor active peptides from the East Asian scorpion. First, dried East Asian scorpion body is pulverized into powder and thoroughly defatted with petroleum ether. Then, the defatted powder is extracted at low temperature with sodium chloride + phosphate buffer solution and stirred. After centrifugation, the supernatant is dialyzed and freeze-dried to obtain a crude protein extract. Subsequently, guided by sample activity, DEAE-52 cation exchange chromatography and HW-40F size exclusion chromatography are used to obtain East Asian scorpion peptide components with antitumor activity. This method reduces extraction costs, improves safety, and offers advantages such as high extraction rate, stable properties, high purity, short extraction time, and high reproducibility. It can be used in the fields of antitumor drugs and health products, and will also provide strong scientific evidence for the development of drugs for treating tumor diseases and the sustainable utilization of natural resources.

[0008] The present invention discloses a method for isolating antitumor active polypeptides from the East Asian scorpion, wherein the East Asian scorpion polypeptide has a molecular weight of 6.5 kDa-10.6 kDa and is a slightly acidic polypeptide. The specific operation is carried out according to the following steps: a. Take the dried body of the East Asian scorpion and crush it into a fine powder. Add petroleum ether to the dried scorpion powder at a mass / volume ratio of 1:5. Shake at room temperature for 4 hours to degrease 3-4 times until colorless. Then, let the degreased East Asian scorpion powder settle naturally and separate from the petroleum ether. Pour off the upper layer of petroleum ether for recovery. Combine the precipitates and dry them naturally to obtain degreased East Asian scorpion powder. Store it in a refrigerator at -40℃ for later use. b. At a material-to-liquid ratio of 1:10, soak the defatted powder from step a in a 0.1 mol / L phosphate buffer solution containing 0.15 mol / L NaCl at a pH of 7.4-7.8 for 4 hours at a temperature of 4°C. Extract the powder three times at a uniform speed of 100 r / min for 12 hours each time to obtain the extract. c. Centrifuge the extract from step b at 7000 r / min and 4℃ for 15 min, then dialyze it at 4℃ in a 3000 Da dialysis bag for 48 hours. Freeze-dry the dialysate for 24 hours at 9.8 Pa and -80℃ to obtain the crude extract. d. Dissolve the crude extract obtained in step c in ultrapure water, add ammonium sulfate to achieve a concentration of 50%, precipitate at 4°C for 12 hours, centrifuge at 10000 r / min at 4°C for 10 minutes to obtain a 50% precipitate fraction; add more ammonium sulfate to the supernatant to increase the concentration to 80%, precipitate at 4°C for 12 hours, centrifuge at 10000 r / min at 4°C for 10 minutes to obtain an 80% precipitate fraction; freeze-dry the obtained fraction for 24 hours at 9.8 Pa and -80°C to obtain 50% and 80% crude protein precipitates; e. Dissolve the 80% crude protein precipitate obtained in step d in 15mM phosphate buffer (pH 6.3). Equilibrate the DEAE-52 anion exchange resin with 15mM phosphate buffer (pH 6.3) at a flow rate of 1mL / min. Perform gradient elution with 0-0.8mol / L sodium chloride and 15mM phosphate buffer. Measure the absorbance at 280 nm and collect the eluent based on the absorption peak to obtain a mixture of four protein and polypeptide components, designated as S1, S2, S3, and S4. f. The mixture of the four protein and polypeptide components from step e is dialyzed and desalted using a 3000 Da dialysis bag at a temperature of 4°C. The dialysis solution is changed every 4 hours for a total of 7 times. Then, it is dried using a vacuum freeze dryer at a temperature of -80°C, a pressure of 10 Pa, and a time of 24 hours to obtain dried powders of the four protein and polypeptide components S1, S2, S3, and S4. g. Dissolve the S4 fraction powder obtained in step f in a 0.1% formic acid-water solution, shake for 10 min, let stand at 4℃ for 1 hour, centrifuge at 4℃ for 10 min and 8000 r / min, and take the supernatant. h. Separate the supernatant obtained in step g using an HW-40F size exclusion column equilibrated with 0.1% formic acid-water solution. The sample loading amount is 50-200 mg, the flow rate is 0.4 mL / min, and the eluent is 0.1% formic acid-water. Four East Asian scorpion polypeptide fractions are obtained and named S4-F1, S4-F2, S4-F3 and S4-F4, respectively.

[0009] The use of S4-F1 in the East Asian scorpion polypeptide fraction obtained by the method in the preparation of the anti-tumor HCT-116 colon cancer drug.

[0010] This invention discloses a method for isolating antitumor active peptides from the East Asian scorpion. The method uses the entire East Asian scorpion as the research object. First, the sample is pulverized at low temperature, defatted 4-6 times with petroleum ether, and extracted three times at 4°C with 0.15 mM NaCl-phosphate buffer for 12 hours each time. The extract is concentrated at low temperature to a certain volume and then precipitated with ammonium sulfate (80%). The precipitate is dissolved in water and filtered through a dialysis bag with a molecular weight of 3000 Da for dialysis to remove impurities. The solution is then freeze-dried at low temperature to obtain a crude powder. The crude powder is then subjected to pH... In 6.5 mM ammonium acetate buffer, anion exchange chromatography was performed using a DEAE-52 column with a flow rate of 1.0 mL / min. The sample loading was 50-200 mg, followed by dialysis using a 3000 Da dialysis bag to remove impurities, yielding four high-purity East Asian scorpion polypeptide fractions, named S4-F1, S4-F2, S4-F3, and S4-F4, with yields of 5.68%, 4.24%, 2.82%, and 3.89%, respectively. Antitumor activity screening and molecular weight distribution analysis were conducted. The activity results showed that the IC50 of S4-F1 against HCT-116 colon cancer cells was 182 μg / mL ± 2.97. 15% SDS-PAGE and Coomassie Brilliant Blue electrophoresis showed that the S4-F1 fraction had a molecular weight range of 6.5 kDa-10.6 kDa and a total protein content of 81.34%.

[0011] This invention discloses a method and application for isolating antitumor active peptides from the East Asian scorpion. The advantages of this method are: It uses low-temperature extraction, which prevents protein decomposition during the extraction process; the use of a buffer solution mixed with sodium chloride maintains the pH stability of the extract and increases the extraction rate of salt-soluble proteins; the invention uses DEAE-52 ion exchange resin to fully enrich and purify effective components of the same type and properties, while HW-40F size exclusion chromatography purifies peptides using a different principle, resulting in peptide samples with higher purity. The preparation method is easily scalable, highly reproducible, low-cost, and pollution-free.

[0012] The innovation of this invention lies in its improvement of the extraction and purification methods based on the extraction method described in application number 202410891681.9, taking into account the characteristics of proteins. This reduces the number of steps in the experimental process that may affect the structure and activity of the compound, minimizing experimental costs while maximizing the yield of active peptide compounds. The preparation method of this invention is simpler and more practical, and the production process is more rational, providing strong scientific evidence for the development of drugs for treating tumor diseases and the sustainable and rational utilization of Xinjiang East Asian scorpion resources. Attached Figure Description

[0013] Figure 1 The chromatogram of East Asian scorpion purified by DEAE-52 anion exchange chromatography is shown in this invention. Figure 2 The chromatogram of East Asian scorpion separated and purified using HW-40F gel size exclusion chromatography is shown in this invention. Figure 3 This is a 15% SDS-PAGE Coomassie Brilliant Blue electrophoresis image of the four components isolated and purified from the East Asian scorpion of this invention. Figure 4 This is a comparison of the inhibition rates of four components obtained by gel chromatography from the S3 part of the East Asian scorpion against HCT-116 colon cancer cells. DOX is the positive control. Compared with the negative control, **p<0.01, ***p<0.001, n=3. Figure 5 The figure represents the inhibition rate of the S4-F1 polypeptide component against HCT-116 colon cancer cells at different concentrations in this invention. Detailed Implementation

[0014] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, the uses and purposes of these exemplary embodiments are only for illustrating the present invention and do not constitute any limitation on the actual protection scope of the present invention, nor are they intended to limit the protection scope of the present invention to this. Example 1

[0015] a. Take 100g of dried East Asian scorpion body, crush it into fine powder, and add petroleum ether to the dried scorpion body fine powder at a mass / volume ratio of 1:5. Shake at room temperature for 4 hours to degrease 3-4 times until colorless. Then, let the degreased East Asian scorpion fine powder settle naturally and separate from the petroleum ether. Pour off the upper layer of petroleum ether for recovery, combine the precipitates, and dry naturally to obtain degreased East Asian scorpion powder. Store in a refrigerator at -40℃ for later use. b. Soak the defatted powder from step a in a 0.1 mol / L phosphate buffer solution containing 0.15 mol / L NaCl at pH 7.4-7.8 for 4 hours at a material-to-liquid ratio of 1:10 for 4 hours at 4°C. Extract the powder three times at a constant speed of 100 r / min for 12 hours each time to obtain the extract. c. Centrifuge the extract from step b at 7000 r / min and 4℃ for 15 min, then dialyze it at 4℃ in a 3000 Da dialysis bag for 48 hours. Freeze-dry the dialysate for 24 hours at 9.8 Pa and -80℃ to obtain the crude extract. d. Dissolve the crude extract obtained in step c in ultrapure water, add ammonium sulfate to achieve a concentration of 50%, precipitate at 4°C for 12 hours, centrifuge at 10000 r / min for 10 minutes at 4°C to obtain a 50% precipitate fraction; add more ammonium sulfate to the supernatant to increase the concentration to 80%, precipitate at 4°C for 12 hours, centrifuge at 10000 r / min for 10 minutes at 4°C to obtain an 80% precipitate fraction; freeze-dry the two precipitates separately for 24 hours at 9.8 Pa and -80°C to obtain 50% and 80% crude protein precipitates; e. Dissolve the 80% crude protein precipitate obtained in step d in 15mM phosphate buffer (pH 6.3). Equilibrate the DEAE-52 anion exchange resin with 15mM phosphate buffer (pH 6.3) at a flow rate of 1mL / min. Perform gradient elution with 0-0.8mol / L sodium chloride and 15mM phosphate buffer. Measure the absorbance at 280 nm and collect the eluent based on the absorption peak to obtain a mixture of four protein and polypeptide components, designated as S1, S2, S3, and S4. f. The mixture of the four protein and polypeptide components from step e is dialyzed and desalted using a 3000 Da dialysis bag at a temperature of 4°C. The dialysis solution is changed every 4 hours for a total of 7 times. Then, it is dried using a vacuum freeze dryer at a temperature of -80°C, a pressure of 10 Pa, and a time of 24 hours to obtain dried powders of the four protein and polypeptide component mixtures S1, S2, S3, and S4. g. Dissolve the S4 fraction powder obtained in step f in a 0.1% formic acid-water solution, shake for 10 min, let stand at 4℃ for 1 hour, centrifuge at 4℃ for 10 min and 8000 r / min, and take the supernatant. h. Separate the supernatant obtained in step g using an HW-40F size exclusion column equilibrated with 0.1% formic acid-water solution. The sample loading amount is 50-200 mg, the flow rate is 0.4 mL / min, and the eluent is 0.1% formic acid-water. Four whole scorpion polypeptide fractions are obtained and named S4-F1, S4-F2, S4-F3, and S4-F4, respectively.

[0016] The high-purity peptide components S4-F1, S4-F2, S4-F3, and S4-F4 obtained in step h were subjected to chemical property detection, SDS-PAGE Coomassie Brilliant Blue electrophoresis, and antitumor activity screening. Example 2

[0017] a. Take 100g of dried East Asian scorpion body, crush it into fine powder, and add petroleum ether to the dried scorpion body fine powder at a mass / volume ratio of 1:5. Shake at room temperature for 4 hours to degrease 3-4 times until colorless. Then, let the degreased East Asian scorpion fine powder settle naturally and separate from the petroleum ether. Pour off the upper layer of petroleum ether for recovery, combine the precipitates, and dry naturally to obtain degreased East Asian scorpion powder. Store in a refrigerator at -40℃ for later use. b. At a material-to-liquid ratio of 1:10, soak the defatted powder from step a in a 0.1 mol / L phosphate buffer solution containing 0.15 mol / L NaCl at a pH of 7.4-7.8 for 4 hours at a temperature of 4°C. Extract the powder three times at a uniform speed of 100 r / min for 12 hours each time to obtain the extract. c. Centrifuge the extract from step b at 7000 r / min and 4℃ for 15 min, then dialyze it at 4℃ in a 3000 Da dialysis bag for 48 hours. Freeze-dry the dialysate for 24 hours at 9.8 Pa and -80℃ to obtain the crude extract. d. Dissolve the crude extract obtained in step c in ultrapure water, add ammonium sulfate to make the concentration reach 50%, precipitate at 4℃ for 12 hours, centrifuge at 10000 r / min for 10 min at 4℃ to obtain a 50% precipitate fraction. Add more ammonium sulfate to the supernatant to increase the concentration to 80%, precipitate at 4℃ for 12 hours, centrifuge at 10000 r / min for 10 min at 4℃ to obtain an 80% precipitate fraction. Then freeze-dry the two precipitates separately for 24 hours at 9.8 Pa and -80℃ to obtain 50% and 80% crude protein precipitates. e. Dissolve 80% of the crude protein precipitate obtained in step d in 15mM phosphate buffer at pH 6.3. Equilibrate the DEAE-52 anion exchange resin with 15mM phosphate buffer at pH 6.3. Elute with a gradient of 0-0.8mol / L sodium chloride and 15mM phosphate buffer at a flow rate of 1mL / min. Measure the absorbance at 280 nm and collect the eluent based on the absorption peak to obtain a mixture of four protein and polypeptide components, namely S1, S2, S3 and S4. f. The mixture of the four protein and polypeptide components from step e is dialyzed and desalted using a 3000 Da dialysis bag at a temperature of 4°C. The dialysis solution is changed every 4 hours for a total of 7 times. Then, it is dried using a vacuum freeze dryer at a temperature of -80°C, a pressure of 10 Pa, and a time of 24 hours to obtain dried powders of the four protein and polypeptide component mixtures S1, S2, S3, and S4. g. Dissolve the S4 fraction powder obtained in step f in a 0.1% formic acid-water solution, shake for 10 min, let stand at 4℃ for 1 hour, centrifuge at 4℃ for 10 min and 8000 r / min, and take the supernatant. h. Separate the supernatant obtained in step g using an HW-40F size exclusion column equilibrated with 0.1% formic acid-water solution. The sample loading amount is 100 mg, the flow rate is 0.4 mL / min, and the eluent is 0.1% formic acid-water. Four whole scorpion polypeptide fractions are obtained and named S4-F1, S4-F2, S4-F3, and S4-F4, respectively. The high-purity peptide components S4-F1, S4-F2, S4-F3, and S4-F4 obtained in step h were subjected to chemical property detection, SDS-PAGE Coomassie Brilliant Blue electrophoresis, and antitumor activity screening. Example 3

[0018] a. Take 100g of dried East Asian scorpion body, crush it into fine powder, and add petroleum ether to the dried scorpion body fine powder at a mass / volume ratio of 1:5. Shake at room temperature for 4 hours to degrease 3-4 times until colorless. Then, let the degreased East Asian scorpion fine powder settle naturally and separate from the petroleum ether. Pour off the upper layer of petroleum ether for recovery, combine the precipitates, and dry naturally to obtain degreased East Asian scorpion powder. Store in a refrigerator at -40℃ for later use. b. Soak the defatted powder from step a in a 0.1 mol / L phosphate buffer solution containing 0.15 mol / L NaCl at a pH of 7.4-7.8 for 4 hours at a temperature of 4°C. Extract the powder three times at a constant speed of 100 r / min for 12 hours each time to obtain the extract. c. Centrifuge the extract from step b at 7000 r / min and 4℃ for 15 min, then dialyze it at 4℃ in a 3000 Da dialysis bag for 48 hours. Freeze-dry the dialysate for 24 hours at 9.8 Pa and -80℃ to obtain the crude extract. d. Dissolve the crude extract obtained in step c in ultrapure water, add ammonium sulfate to achieve a concentration of 50%, precipitate at 4°C for 12 hours, centrifuge at 10000 r / min for 10 minutes at 4°C to obtain a 50% precipitate; add more ammonium sulfate to the supernatant to increase the concentration to 80%, precipitate at 4°C for 12 hours, centrifuge at 10000 r / min for 10 minutes at 4°C to obtain an 80% precipitate; freeze-dry the two precipitates separately for 24 hours at 9.8 Pa and -80°C to obtain 50% and 80% crude protein precipitates, respectively. e. Dissolve the 80% crude protein precipitate obtained in step d in 15mM phosphate buffer (pH 6.3). Equilibrate the DEAE-52 anion exchange resin with 15mM phosphate buffer (pH 6.3) at a flow rate of 1mL / min. Perform gradient elution with 0-0.8mol / L sodium chloride and 15mM phosphate buffer. Measure the absorbance at 280 nm and collect the eluent based on the absorption peak to obtain a mixture of four protein and polypeptide components, designated as S1, S2, S3, and S4. f. The mixture of the four protein and polypeptide components from step e is dialyzed and desalted using a 3000 Da dialysis bag at a temperature of 4°C. The dialysis solution is changed every 4 hours for a total of 7 times. Then, it is dried using a vacuum freeze dryer at a temperature of -80°C, a pressure of 10 Pa, and a time of 24 hours to obtain dried powders of the four protein and polypeptide component mixtures S1, S2, S3, and S4. g. Dissolve the S4 fraction powder obtained in step f in a 0.1% formic acid-water solution, shake for 10 min, let stand at 4℃ for 1 hour, centrifuge at 4℃ for 10 min and 8000 r / min, and take the supernatant. h. Separate the supernatant obtained in step g using an HW-40F size exclusion column equilibrated with 0.1% formic acid-water solution. The sample loading amount is 150 mg, the flow rate is 0.4 mL / min, and the eluent is 0.1% formic acid-water. Four whole scorpion polypeptide fractions are obtained and named S4-F1, S4-F2, S4-F3, and S4-F4, respectively.

[0019] The high-purity peptide components S4-F1, S4-F2, S4-F3, and S4-F4 obtained in step h were subjected to chemical property detection, SDS-PAGE Coomassie Brilliant Blue electrophoresis, and antitumor activity screening. Example 4

[0020] a. Take 100g of dried East Asian scorpion body, crush it into fine powder, and add petroleum ether to the dried scorpion body fine powder at a mass / volume ratio of 1:5. Shake at room temperature for 4 hours to degrease 3-4 times until colorless. Then, let the degreased East Asian scorpion fine powder settle naturally and separate from the petroleum ether. Pour off the upper layer of petroleum ether for recovery, combine the precipitates, and dry naturally to obtain degreased East Asian scorpion powder. Store in a refrigerator at -40℃ for later use. b. At a material-to-liquid ratio of 1:10, soak the defatted powder from step a in a 0.1 mol / L phosphate buffer solution containing 0.15 mol / L NaCl at pH 7.4-7.8 for 4 hours at 4°C. Extract the powder three times at a constant speed of 100 r / min for 12 hours each time to obtain the extract. c. Centrifuge the extract from step b at 7000 r / min and 4℃ for 15 min, then dialyze it at 4℃ in a 3000 Da dialysis bag for 48 hours. Freeze-dry the dialysate for 24 hours at 9.8 Pa and -80℃ to obtain the crude extract. d. Dissolve the crude extract obtained in step c in ultrapure water, add ammonium sulfate to make the concentration reach 50%, precipitate at 4℃ for 12 hours, centrifuge at 10000 r / min for 10 min at 4℃ to obtain a 50% precipitate fraction. Add more ammonium sulfate to the supernatant to increase the concentration to 80%, precipitate at 4℃ for 12 hours, centrifuge at 10000 r / min for 10 min at 4℃ to obtain an 80% precipitate fraction. Then freeze-dry the two precipitates separately for 24 hours at 9.8 Pa and -80℃ to obtain 50% and 80% crude protein precipitates. e. Dissolve the 80% protein precipitate obtained in step d in 15mM phosphate buffer (pH 6.3). Equilibrate the DEAE-52 anion exchange resin with 15mM phosphate buffer (pH 6.3) at a flow rate of 1mL / min. Perform gradient elution with 0-0.8mol / L sodium chloride and 15mM phosphate buffer. Measure the absorbance at 280 nm and collect the eluent based on the absorption peak to obtain a mixture of four protein and polypeptide components, designated as S1, S2, S3, and S4. f. The mixture of the four protein and polypeptide components from step e is dialyzed and desalted using a 3000 Da dialysis bag at a temperature of 4°C. The dialysis solution is changed every 4 hours for a total of 7 times. Then, it is dried using a vacuum freeze dryer at a temperature of -80°C, a pressure of 10 Pa, and a time of 24 hours to obtain dried powders of the four protein and polypeptide component mixtures S1, S2, S3, and S4. g. Dissolve the S4 fraction powder obtained in step f in a 0.1% formic acid-water solution, shake for 10 min, let stand at 4℃ for 1 hour, centrifuge at 4℃ for 10 min and 8000 r / min, and take the supernatant. h. Separate the supernatant obtained in step g using an HW-40F size exclusion column equilibrated with 0.1% formic acid-water solution. The sample loading amount is 200 mg, the flow rate is 0.4 mL / min, and the eluent is 0.1% formic acid-water. Four whole scorpion polypeptide fractions are obtained and named S4-F1, S4-F2, S4-F3, and S4-F4, respectively.

[0021] The high-purity peptide components S4-F1, S4-F2, S4-F3, and S4-F4 obtained in step h were subjected to chemical property detection, SDS-PAGE Coomassie Brilliant Blue electrophoresis, and antitumor activity screening. Example 5

[0022] The contents of the high-purity polypeptide components obtained in Examples 1-4 were determined and are shown in Table 1: Experimental methods: Protein content was detected using a BCA assay kit, with bovine serum albumin (BSA) as the standard curve. Standard protein solutions were prepared at concentrations of 1.2 mg / mL, 1 mg / mL, 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL, 0.2 mg / mL, and 0 mg / mL. To ensure the sample protein concentration fell as close as possible to the midpoint of the standard curve, the sample concentration was set to 2 mg / mL. Working solution A and working solution B were mixed at a 50:1 ratio according to the required volume of working solution, shaken well, and stored in the dark (prepare immediately before use). 100 μL of protein solution (standard solution and test protein solution) and 800 μL of the working solution were added to a 1.5 mL centrifuge tube. Each experiment was repeated in triplicate. After reacting at 37°C for 30 minutes in a benchtop drying oven, 250 μL of the reaction mixture was transferred to a 96-well plate and analyzed using a microplate reader at a wavelength of 562 nm. The absorbance was measured at nm. A standard curve was plotted with the protein concentration and absorbance of the standard as independent and dependent variables, respectively. The protein concentration (mg / mL) of the sample was obtained by substituting the absorbance of the sample into the standard curve. The protein content of the sample was obtained by formula 1. Formula 1 Note: C1 is the protein concentration of the sample calculated by substituting the sample into the standard curve, and C0 is the concentration of the sample being tested. Table 1. Protein content of four high-purity components of the East Asian pincer scorpion.

[0023] As can be seen from Table 1, the protein content of S4-F1 in the four high-purity components is over 80%. Example 6

[0024] The molecular weight of the high-purity polypeptide components obtained in Examples 1-4 was estimated. Experimental methods: The molecular weights of four high-purity peptide fractions were estimated using sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). Using a 5% stacking gel and a 15% separating gel, 20 μL of protein sample and 4 μL of molecular weight marker were added to different lanes of the gel under non-reducing conditions for analysis. The stacking gel was run at 75 V, and the separating gel at 120 V, until the sample reached the bottom of the separating gel, and then stained with Coomassie blue. The molecular weights of the three proteins S1, S2, and S3 were estimated by comparing them with the molecular weight marker bands. Example 7

[0025] The high-purity polypeptide components obtained in Examples 1-4 were selected for antitumor activity screening to detect HCT-116 cell inhibitory activity: The sample was prepared into a 0.5 mg / mL protein solution using cell culture medium. Cells in the logarithmic growth phase were digested and resuspended, and seeded at the appropriate cell density in 96-well plates. 100 μL of cell culture was added to each well. The cells were cultured for 24 hours in a cell culture incubator at 37℃, 5% CO2, and 95% humidity. 20 μL of the test compound was added, the supernatant was discarded, and 100 μL of protein solution of different concentrations was added. 3-6 replicates were set up, and a cell-free background group, a solvent control group, and a positive control group were also included. After culturing for another 48 hours at 37℃ and 5% CO2, the supernatant was discarded, and 100 μL / well thiazolyl blue solution was added. The cells were cultured for another 2-4 hours. The supernatant was discarded, and 150 μL of DMSO was added to each well, and the cells were shaken slowly for 10 min to dissolve the crystals. After the formazan dissolved, the absorbance (OD value) at 570 nm was measured using a microplate reader. The inhibition rate of the compound on tumor cell growth was calculated using Formula 2. The results are shown in Table 2. Formula 2 Table 2. HCT-116 cell inhibition rates of four high-purity components from the East Asian pincer scorpion.

[0026] The results showed that among the four high-purity polypeptide components, the S4-F1 component had the highest inhibition rate against HCT-116 at 91.13%, and the higher the protein content of the sample, the stronger the inhibition rate against tumor cells. Example 8

[0027] The high-purity polypeptide fraction S4-F1 obtained in Examples 1-4 was identified using LC-MS / MS: The ZipTip C18 method was used for desalting, and the specific steps are as follows: ① Take the sample and dissolve it thoroughly with 0.1% TFA; ② Rinse the tip 10 times with 50 μl of 60% ACN / 0.1% TFA; ③ Wash the tip 10 times with 10 μl of 0.1% TFA; ④ Aspirate and expel the sample from the tip 20 times, and drain the liquid; ⑤ Wash the tip 5 times with 10 μl of 0.1% TFA; ⑥ Elute the peptides into a new EP tube with 10 μl of 60% ACN and 0.1% TFA, and vacuum dry; Dissolve the thoroughly desalted sample in 10 μL of dissolving buffer (0.1% formic acid), vortex thoroughly, centrifuge at 17000 rpm and 4℃ for 20 min, transfer the supernatant to a sample tube, and take 3 μL for capillary high-performance liquid chromatography (HPLC: Thermo EASY nLC 1000, column: 75 μm x 1000). 150 mm, PepMap RSLC C18, 2 μm, 100 Å); Chromatographic conditions: Phase A: 0.1% formic acid, 5% ACN; Phase B: 0.1% formic acid, ACN, using a gradient elution program: 0-3 min, 0-4% B; 3-45 min, 4-20% B; 45-51 min, 20-32% B; 51-52 min, 32-90% B; 52-60 min, 90-90% B; The separated peptide products were analyzed by mass spectrometry using a Thermo Scientific QE mass spectrometer for 60 min. Detection mode: positive ion. The mass-charge ratio of the peptide and peptide fragments was determined by full scan. Ten fragment spectra were acquired, and PEAKS software was used for database searching. Proteins with the same characteristic peptides as those detected by mass spectrometry were searched in the Uniprot database using Scorpion as the species limit. The final results of protein identification and quantification are shown in Table 3. Table 3. LC-MS / MS peptide identification results of East Asian scorpion S4-F1 peptide components

[0028] The results showed that the molecular weight of the polypeptide components in the S4-F1 region of the East Asian scorpion ranged from 4.33 to 10.43, which was basically consistent with the experimental results of SDS-PAG. Among them, the identified polypeptide components consisted of toxic peptides and antimicrobial peptides, and possessed antibacterial, ion channel interference, enzyme inhibition, membrane disruption, and immunomodulatory biological activities related to antitumor activity.

[0029] The method described in this invention is simpler and more practical, and the production process is more rational, providing strong scientific evidence for the development of drugs for treating colorectal cancer and the sustainable and rational utilization of Xinjiang scorpion resources. The preparation method of this invention is simpler and more practical, and the production process is more rational, providing strong scientific evidence for the development of drugs for treating colorectal cancer and the sustainable utilization of scorpion resources.

[0030] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for isolating antitumor active polypeptides from East Asian scorpion, characterized in that, The East Asian scorpion polypeptide has a molecular weight of 6.5kDa-10.6kDa; it is a slightly acidic polypeptide, and the specific operation is carried out according to the following steps: a. Take the dried body of the East Asian scorpion and crush it into a fine powder. Add petroleum ether to the dried scorpion powder at a mass / volume ratio of 1:

5. Shake at room temperature for 4 hours to degrease 3-4 times until colorless. Then, let the degreased East Asian scorpion powder settle naturally and separate from the petroleum ether. Pour off the upper layer of petroleum ether for recovery. Combine the precipitates and dry them naturally to obtain degreased East Asian scorpion powder. Store it in a refrigerator at -40℃ for later use. b. At a material-to-liquid ratio of 1:10, soak the defatted powder from step a in a 0.1 mol / L phosphate buffer solution containing 0.15 mol / L NaCl at a pH of 7.4-7.8 for 4 hours at a temperature of 4°C. Extract the powder three times at a uniform speed of 100 r / min for 12 hours each time to obtain the extract. c. Centrifuge the extract from step b at 7000 r / min and 4℃ for 15 min, then dialyze it at 4℃ in a 3000 Da dialysis bag for 48 hours. Freeze-dry the dialysate for 24 hours at 9.8 Pa and -80℃ to obtain the crude extract. d. Dissolve the crude extract obtained in step c in ultrapure water, add ammonium sulfate to achieve a concentration of 50%, precipitate at 4°C for 12 hours, centrifuge at 10000 r / min at 4°C for 10 minutes to obtain a 50% precipitate fraction; add more ammonium sulfate to the supernatant to increase the concentration to 80%, precipitate at 4°C for 12 hours, centrifuge at 10000 r / min at 4°C for 10 minutes to obtain an 80% precipitate fraction; freeze-dry the obtained fraction for 24 hours at 9.8 Pa and -80°C to obtain 50% and 80% crude protein precipitates; e. Dissolve the 80% crude protein precipitate obtained in step d in 15mM phosphate buffer (pH 6.3). Equilibrate the DEAE-52 anion exchange resin with 15mM phosphate buffer (pH 6.3) at a flow rate of 1mL / min. Perform gradient elution with 0-0.8mol / L sodium chloride and 15mM phosphate buffer. Measure the absorbance at 280 nm and collect the eluent based on the absorption peak to obtain a mixture of four protein and polypeptide components, designated as S1, S2, S3, and S4. f. The mixture of the four protein and polypeptide components from step e is dialyzed and desalted using a 3000 Da dialysis bag at a temperature of 4°C. The dialysis solution is changed every 4 hours for a total of 7 times. Then, it is dried using a vacuum freeze dryer at a temperature of -80°C, a pressure of 10 Pa, and a time of 24 hours to obtain dried powders of the four protein and polypeptide components S1, S2, S3, and S4. g. Dissolve the S4 fraction powder obtained in step f in a 0.1% formic acid-water solution, shake for 10 min, let stand at 4℃ for 1 hour, centrifuge at 4℃ for 10 min and 8000 r / min, and take the supernatant. h. Separate the supernatant obtained in step g using an HW-40F size exclusion column equilibrated with 0.1% formic acid-water solution. The sample loading amount is 50-200 mg, the flow rate is 0.4 mL / min, and the eluent is 0.1% formic acid-water. Four East Asian scorpion polypeptide fractions are obtained and named S4-F1, S4-F2, S4-F3 and S4-F4, respectively.

2. The use of S4-F1 in the East Asian scorpion polypeptide fraction obtained by the method of claim 1 in the preparation of the antitumor HCT-116 colon cancer drug.

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