Extraction process and activity application of corydalus cornutus polypeptide

Abstract

The invention discloses a corydalus cornutus polypeptide extraction process and activity application, which comprises the following steps: extracting by adopting an enzymolysis method to break peptide bonds of proteins into smaller peptide fragments, salting out to obtain crude polypeptide, further separating by adopting sephadex G-250, carrying out electrophoretic analysis by adopting Tris-tricine-SDS-PAGE, taking a target component, and carrying out semi-preparative liquid phase purification to obtain the corydalus cornutus polypeptide. After a polypeptide sequence is identified by a liquid chromatograph-mass spectrometer, several polypeptides with extremely strong activity are screened by combining an online tool and molecular docking, and a standard substance is synthesized to verify the activity of the polypeptides. According to the invention, the corydalus cornutus is subjected to polypeptide extraction and separation for the first time, four kinds of corydalus cornutus polypeptides with extremely strong activity are identified, in-vitro activity evaluation analysis finds that the corydalus cornutus polypeptides can significantly relieve the oxidative stress state of HK-2 cells, which indicates that the corydalus cornutus polypeptides have a good kidney protection effect, and a theoretical basis can be provided for development and application of the corydalus cornutus.

Description

Technical Field

[0001] The present invention relates to biological polypeptides, extraction processes and applications thereof, and particularly to polypeptides extracted from Corydalus larvae as raw materials. Background Art

[0002] Corydalus larvae is a general term for the larvae of insects in the family Corydalidae, order Megaloptera. It has the effects of tonifying qi and kidney, dispelling dampness and relieving pain, and strengthening the body resistance. It is mainly used in folk medicine to treat enuresis in children and frequent micturition in the elderly. Modern research on the pharmacodynamic material basis has found that Corydalus larvae is rich in protein, and the water-soluble protein accounts for 4.87%. As a kind of insect that is both a medicine and a food commonly used in the folk in special regions, Corydalus larvae has extremely high nutritional value. However, through literature review, it is found that there is less research on the components of Corydalus larvae at present, and the quality research on Corydalus larvae is not comprehensive enough. Therefore, it is necessary to conduct in-depth research on the pharmacodynamic material basis of Corydalus larvae.

[0003] Polypeptides are one of the main pharmacodynamic components in animal drugs. Their molecular weight is generally less than 10 kDa, and they have functions such as anti-inflammatory, anti-tumor, antibacterial, immune regulation, and liver and kidney protection. Natural active polypeptides widely exist in nature. They are molecular polymers composed of 2 - 20 amino acids, are easily digested and absorbed by the human body, and have the characteristics of high safety, strong biological activity, and high target specificity. LC-MS / MS is a highly sensitive and selective method with powerful separation and identification capabilities. It is an effective means for analyzing complex samples and is widely used in the identification of various macromolecules and small molecules. As an analysis tool for bioactive peptides, it can perform sequence analysis on a large number of polypeptide fragments, improving the accuracy of polypeptide sequence identification. Computer simulation prediction is widely used in the research of polypeptide activity screening. By using online tools to predict the biological activity and toxicity of polypeptides, the research cycle is significantly shortened, the research efficiency and the success rate of active peptide screening are improved, providing an important idea for the discovery of new active polypeptides.

[0004] There is no research report on polypeptides extracted from Corydalus larvae in the prior art. Summary of the Invention

[0005] The purpose of the present invention is to extract polypeptides from Corydalus larvae, and through analysis, screening, prediction and verification, find out its effective components, and develop a drug development and quality control plan using Corydalus larvae as raw materials.

[0006] To achieve the above purpose, the technical solution adopted is as follows: Corydalus larvae polypeptide, with the amino acid sequences: KLGAAPFHFW, IFFLFII, LIRF and LEI.

[0007] Number Sequence (single letter) Amino acid sequence (three letters) PSC-1 KLGAAPFHFW LysLeuGlyAlaAlaProPheHisPheTrp PSC-2 IFFLFII IlePhePheLeuPheIleIle PSC-3 LIRF LeuIleArgPhe PSC-4 LEI LeuGluIle .

[0008] and the extraction process of the above-mentioned dragonfly larvae polypeptide, which comprises the following steps: (1) Using dried dragonfly larvae as raw materials, extracting polypeptides by enzymatic hydrolysis, and taking the enzymatic hydrolysate supernatant after centrifugation; (2) Adding alginic acid to the enzymatic hydrolysate supernatant for adsorption and then eluting to obtain a polypeptide solution. After adjusting the pH to the isoelectric point, further separating by salting-out method to obtain crude salting-out polypeptides; (3) Further separating the crude salting-out polypeptides by Sephadex G-50 dextran gel, detecting the absorbance at 280 nm with an ultraviolet detector, preparing an elution curve, and freeze-drying after combining the elution peaks to obtain crude polypeptides; (4) Using Tris-tricine-SDS-PAGE electrophoresis to detect whether there are polypeptides in the elution components, and subjecting the components containing crude polypeptides to reverse-phase preparative purification to remove small molecule impurities in the dragonfly larvae polypeptides, and freeze-drying the cut peak segments to obtain dragonfly larvae polypeptides.

[0009] Further, in the step (1), after the dragonfly larvae raw materials are crushed, they are defatted by petroleum ether using a Soxhlet extractor, and then the subsequent extraction is carried out.

[0010] Further, in the step (1), taking dragonfly larvae powder in a conical flask, adding distilled water to adjust the substrate concentration to 30 mg / mL, adding 1 mol / L HCl to adjust the pH of the solution to 2.0, adding pepsin, hydrolyzing at 37°C for 1 h, performing a water bath at 80°C for 10 min to inactivate the protease, and centrifuging at 3500 rpm for 15 min to obtain the enzymatic hydrolysate supernatant.

[0011] Further, in the step (2), After adding alginic acid to the enzymatic hydrolysate supernatant, stir for 30 min, then place it in a refrigerator at 4°C and let it stand for 30 min, filter by suction to obtain alginic acid, and elute with 0.2 mol / L HCl to obtain a polypeptide solution; after adjusting the pH to 3.5 with sodium acetate, add sodium chloride to a 10% saturation state, place it in a refrigerator at 4°C for overnight salting-out, centrifuge to collect the precipitate, and freeze-dry to obtain crude salting-out polypeptides.

[0012] Further, the 10% saturation state means adding 3.2 g of sodium chloride to every 100 mL of the enzymatic hydrolysis solution.

[0013] Further, after Sephadex G-50 is swollen, it is loaded into a chromatography column. The crude salting-out polypeptides are dissolved in deionized water, filtered through a 0.45 μm filter membrane, the sample is slowly added into the chromatography column, and eluted with deionized water; collected regularly with an automatic collector and labeled with a receiving tube.

[0014] Further, the chromatographic conditions for the reverse-phase preparation are as follows: JADE-PAK ODS AQ C18 chromatographic column, 250 × 10 mm, 5 μm, the sample preparation concentration is 40 mg / mL, the flow rate is 2 mL / min, and the injection volume is 50 μL.

[0015] And the application of the above-mentioned Polypedilum vanderplanki polypeptide, the polypeptide is used for preparing a drug with a renal protection effect.

[0016] And the application of the above-mentioned Polypedilum vanderplanki polypeptide, the polypeptide is used for preparing a health product with a renal protection effect.

[0017] The beneficial effects of adopting the above scheme are as follows: The present invention extracts natural polypeptides from Polypedilum vanderplanki as raw materials. After enzymatic hydrolysis, the obtained components are subjected to salting out, Sephadex G-50 gel column chromatography, and reverse-phase liquid-phase preparation. After analyzing them by LC-MS, a total of 52 polypeptides are detected. After respectively performing activity prediction and toxicity prediction through Peptide Ranker and ToxinPred, 12 polypeptides with an activity score > 0.5 and no toxicity are screened out. On this basis, active polypeptides with an activity score > 0.8 and relatively high-content peptide segments: KLGAAPFHFW, IFFLFII, LIRF, and LEI are further screened out, and are respectively named PSC-2-1, PSC-2-2, PSC-2-3, and PSC-2-4. Further molecular docking reveals that the Polypedilum vanderplanki polypeptides are respectively docked with Keap-1 and Nrf2-related proteins, and the binding energy is less than -5.0 kcal / mol, indicating that the 4 peptide segments have a strong antioxidant stress effect. Through in vitro verification experiments, it is found that the polypeptide has a significant renal protection effect. The present invention provides an extraction process for extracting and separating natural polypeptides from Polypedilum vanderplanki by combining enzymatic hydrolysis, salting out, column chromatography, and liquid-phase preparation. After sequence identification by LC-MS / MS, by comprehensively applying the methods of Peptide Ranker, ToxinPred, molecular docking, and in vitro verification, active natural polypeptides with a renal protection effect are effectively screened out. The polypeptides screened this time can be used as pharmacodynamic components to further evaluate the quality of Polypedilum vanderplanki, providing a theoretical basis for the development and quality control of Polypedilum vanderplanki. Description of the Drawings

[0018] Figure 1 It is the Sephadex G-50 elution curve of the Polypedilum vanderplanki polypeptide of this application; Figure 2 It is the Tris-tricine-SDS-PAGE electrophoresis diagram in this application; Figure 3 It is the semi-preparative liquid-phase analysis chromatogram of the Polypedilum vanderplanki polypeptide PSC-2 of this application; Figure 4It is the docking diagram of 4 polypeptides in this application with Keap-1 and Nrf2-related protein molecules; Figure 5 It is the screening of the optimal concentration for H2O2-induced HK-2 cell damage in this application; Figure 6 It is the effect of 4 polypeptides in this application on the viability of H2O2-induced HK-2 cells. Specific implementation manners

[0019] Next, in combination with the specific embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. The described embodiments are only a part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts belong to the scope of protection of the present invention.

[0020] 1 Materials and methods 1.1 Instruments Easy-nLC 1200 / QExactive liquid chromatography-mass spectrometer (Thermo Fisher Scientific, USA); SCI-VS vortex mixer (SCILOGEX, USA); DH3600A constant temperature incubator (Tianjin Test Instrument Co., Ltd.); FB2085 one over one hundred thousandth electronic balance (Shanghai Shunyu Hengping Scientific Instrument Co., Ltd.); Devices Spectra Max190 full wavelength microplate reader (Molecular Devices, USA), PowerPacTMBasic electrophoresis system (Bio-Rad, USA), ultraviolet-visible spectrophotometer (Shanghai Yidian Analytical Instrument Co., Ltd.), pH meter (Shanghai Yidian Scientific Instrument Co., Ltd.), automatic collector (Shanghai Qingpu Huxi Instrument Factory), vacuum freeze dryer (Xiniu Technology).

[0021] 1.2 Test drugs Polypeptides PSC-2-1 (KLGAAPFHFW), PSC-2-2 (IFFLFII), PSC-2-3 (LIRF), PSC-2-4 (LEI) of the climbing water beetle were further synthesized by Sangon Biotech (Shanghai) Co., Ltd. (purity > 98%).

[0022] Dried bodies of the climbing water beetle were purchased from the ecological breeding base in Yuxi City, Yunnan Province.

[0023] 1.3 Reagents Alginic acid (Shanghai Yuanye Bio-Technology Co., Ltd.), absolute ethanol (Tianjin Fuyu Fine Chemical Co., Ltd.), acetic acid (Chengdu Jinshan Chemical Reagent Co., Ltd.), sodium chloride (analytical pure, Chengdu Jinshan Chemical Reagent Co., Ltd.), sodium acetate (Chengdu Jinshan Chemical Reagent Co., Ltd.), Sephadex G-50 (Beijing Solarbio Science & Technology Co., Ltd.), hydrochloric acid (analytical pure, Chongqing Chuandong Chemical (Group) Co., Ltd.), pepsin 1:10000 (Beijing Solarbio Science & Technology Co., Ltd.).

[0024] Example 1: Extraction, isolation and purification of the polypeptides from the larvae of Neoperla (1) The larvae of Neoperla were crushed and defatted with petroleum ether using a Soxhlet extractor, and then air-dried. The defatted and dried larval powder was placed in a conical flask, and distilled water was added to adjust the substrate concentration to 30 mg / mL. The pH of the solution was adjusted to 2.0 with 1 mol / L HCl, and pepsin was added. Hydrolysis was carried out at 37 °C for 1 h, followed by a 10-min water bath at 80 °C to inactivate the protease. The mixture was centrifuged at 3500 rpm for 15 min, and the supernatant was collected to obtain the enzymatic hydrolysate. A certain amount of alginic acid was added, and after stirring for 30 min, the mixture was placed in a refrigerator at 4 °C for 30 min. The alginic acid was filtered by suction, and elution was carried out with 0.2 mol / L HCl to obtain a polypeptide solution.

[0025] (2) The pH of the polypeptide solution was adjusted to 3.5 ± 0.05 with sodium acetate, and sodium chloride was added to a 10% saturated state. The mixture was placed in a refrigerator at 4 °C for overnight salting-out. The precipitate was collected by centrifugation and freeze-dried to obtain crude peptides. (3) Sephadex G-50 was packed into a chromatography column. The freeze-dried crude peptides were dissolved in deionized water and filtered through a 0.45-μm filter membrane. The sample was slowly added into the chromatography column, and elution was carried out with deionized water. An automatic collector was used to collect the eluate at regular intervals, and the receiving tubes were labeled. The absorbance was detected at 280 nm with a UV detector. The elution curve was prepared as shown in Figure 1 , and the elution peaks were combined and freeze-dried to obtain crude polypeptides. Four polypeptide components from the larvae of Neoperla were obtained and named PSC-1, PSC-2, PSC-3, and PSC-4 respectively. (4) Tris-tricine-SDS-PAGE electrophoresis was used to detect whether there were polypeptides in the eluted fractions. The fraction with the most polypeptides and the highest quality was selected for further analysis. The electrophoresis results are shown in Figure 2 as follows; (5) The crude polypeptide was subjected to reversed-phase preparative purification to remove small-molecule impurities in the Polygonum hydropiper polypeptide, and the peak-eluting segment was intercepted and freeze-dried to obtain the Polygonum hydropiper polypeptide; Analytical chromatographic conditions: JADE-PAK ODS AQ C18 chromatographic column (250 × 4.6 mm, 5 μm), detection wavelength: 214 nm; injection volume: 10 μL; mobile phase A: 0.1% TFA water, mobile phase B: 0.06% TFA acetonitrile; flow rate: 1.0 mL / min; gradient elution: 0 - 5 min, 2% - 10% B; 5 - 6 min, 10% - 35% B; 6 - 20 min, 35% - 40% B; 20 - 21 min, 40% - 2% B; 21 - 30 min, 2% - 2% B, and the chromatogram is as shown in Figure 3 shown; (6) Preparation chromatographic conditions: JADE-PAK ODS AQ C18 chromatographic column (250 × 10 mm, 5 μm), flow rate: 2 mL / min, injection volume: 50 μL; other conditions are the same as the analytical conditions.

[0026] (7) The polypeptides obtained from semi-preparation were combined and freeze-dried to obtain the mixed Polygonum hydropiper polypeptide, which was named PSC-2-H and subjected to the next analysis and identification; (8) The Polygonum hydropiper polypeptide PSC-2-H was subjected to LC-MS / MS for polypeptide identification. The liquid chromatography-mass spectrometry (LC-MS / MS) detected a total of 52 polypeptides in this component. The obtained polypeptide sequences were input into the online tool Peptide Ranker for preliminary activity screening, and ToxinPred for toxicity prediction to screen for polypeptides with strong activity and the polypeptide with the highest relative content in the component, and molecular docking was used to predict its antioxidant stress effect.

[0027] The online website Peptide Ranker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to score the Polygonum hydropiper polypeptide sequence, and the score range was 0 - 1. When the score value > 0.5, it indicated that the polypeptide had activity, and when the score > 0.8, it indicated that the polypeptide had strong activity; the online tool ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) was used to evaluate the toxicity of the active polypeptide sequence.

[0028] Polypeptides with an output result score > 0.5 are considered to have activity, and a score > 0.8 is considered to have strong activity. Among them, 12 polypeptides have an activity score > 0.5 and are non-toxic. On this basis, active polypeptides with an activity score > 0.8 and components with relatively high contents were further screened out and imported into ToxinPred for toxicity prediction. The results showed that none of the obtained active peptide segments were toxic, and the specific information of the peptide segments with a prediction score greater than 0.5 is shown in Table 1: Table 1 Prediction results of the biological activity and toxicity of Mylabris polypeptides Polypeptide sequence Length Activity score Toxicity Isolation quantity KLGAAPFHFW 10 0.956659 Non-Toxin 1173.52 IFFLFII 7 0.900088 Non-Toxin 912.29 LIRF 4 0.871055 Non-Toxin 547.75 YSPALL 6 0.726612 Non-Toxin 662.86 KLIF 4 0.689092 Non-Toxin 519.74 LNSLILMTWQKIAPIILISYNYSYS 25 0.666041 Non-Toxin 2945.91 PALL 4 0.6574 Non-Toxin 412.58 VSGSMILAGILKLGGYGLMRVLML 24 0.654729 Non-Toxin 2493.57 LLLLL 5 0.549762 Non-Toxin 583.9 LAYFHLLTHALFKALLFMAGSVIHNM 26 0.529938 Non-Toxin 2960.02 VGAHINVGRTYNHLKMF 17 0.515016 Non-Toxin 1957.55 FDPIIE 6 0.512527 Non-Toxin 732.91 。

[0029] Example 2 Molecular docking of Nepa chinensis Hoff polypeptide The molecular structure of the polypeptide was transformed and constructed through Novopro Biotechnology (https: / / www.novopro.cn / tools / smiles2pdb) to obtain the PDB format. The crystal structures of the Nepa chinensis Hoff polypeptide related to the Keap-1 target (PDB: 4L7B) and the Nrf2-related target (PDB: 7K2L) were obtained from the PDB database. The active polypeptide was subjected to molecular docking using Autodock-vina software and visualized using pymol software, as Figure 4 shown. The molecular docking results are shown in Table 2. The binding energies are all less than -5.0 kcal / mol, indicating that the Nepa chinensis Hoff polypeptides PSC-2-1, PSC-2-2, PSC-2-3, and PSC-2-4 bind well to the renal protection-related targets. Therefore, the 4 polypeptides were further synthesized to study their in vitro biological activities.

[0030] Table 2 Molecular docking results of Nepa chinensis Hoff polypeptides Protein Compound Binding energy (kcal / mol) 4L7B PSC-2-1 -9.2 4L7B PSC-2-2 -8.7 4L7B PSC-2-3 -8.2 4L7B PSC-2-4 -7.7 7K2L PSC-2-1 -9 7K2L PSC-2-2 -8.1 7K2L PSC-2-3 -8.3 7K2L PSC-2-4 -7.6 。

[0031] Example 3 Study on the in vitro protective activity of Nepa chinensis Hoff polypeptide against H2O2-induced damage to renal HK-2 cells (1) Culture of human renal proximal tubular epithelial HK-2 cells: HK-2 cells were placed in complete medium and statically cultured in an incubator at 37 °C, 5% CO2, and a relative humidity of 95%. When the cell confluence reached 80% - 90%, subculture was carried out, and cells in good growth condition and in the logarithmic growth phase were taken for the experiment.

[0032] (2) Screening for the optimal concentration of H2O2 HK-2 cells were seeded at 1.5×10 4Cells at a density of

[0033] The cells were seeded into 96-well plates at a density of Figure 5 cells / well. After the cells adhered under the culture conditions of 37 °C and 5% CO2, the cells were divided into a zero-adjustment group, a control group, and intervention groups with different doses of H2O2 (1000, 900, 800, 700, 600, 500, 400 μM), with 5 replicate wells in each group. After 24 h of culture, the cells were rinsed 3 times with PBS at room temperature. 100 μL of basal medium was added to the blank control group and the model group, and polypeptide solutions with final concentrations of 1000, 900, 800, 700, 600, 500, and 400 μM were added to the intervention groups with different doses of H2O2, respectively. After the reaction was terminated after 2 h of intervention, 10 μL of CCK-8 was added to each well, and the cells were further cultured at 37 °C for 60 min. The absorbance value (OD) was measured at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability was calculated. P <0.01), indicating an obvious cell damage effect. Therefore, a H2O2 injury model was established by treating HK-2 cells with 500 μM H2O2 for 2 h.

[0034] (3) Detection of the effects of Polychaos crawfordi polypeptides (PSC-2-H, PSC-2-1, PSC-2-2, PSC-2-3, PSC-2-4) on the viability of HK-2 cells by the CCK-8 method HK-2 cells were seeded into 96-well plates at a density of 1.5×10 4 cells / well. After the cells adhered under the culture conditions of 37 °C and 5% CO2, the cells were divided into a zero-adjustment group, a blank control group (Control), a model group (Model), and a drug administration group, with 5 replicate wells in each group. After 24 h of culture, the cells were rinsed 3 times with PBS at room temperature. 100 μL of basal medium was added to the blank control group and the model group, and PSC-2-1, PSC-2-3, and PSC-2-4 with final concentrations of 10, 50, and 100 μmol / L, PSC-2-2 with final concentrations of 5, 10, and 25 μmol / L, and PSC-2-H with final concentrations of 100, 50, and 10 μg / L were added to the intervention groups with different doses, respectively, for 6 h of intervention. Then, the medium containing 500 μmol / L H2O2 was replaced for cell modeling for 2 h, and then the reaction was terminated. 10 μL of CCK-8 was added to each well, and the cells were further cultured at 37 °C for 60 min. The absorbance value (OD) was measured at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability was calculated.

[0035] ; Note: As: experimental well; Ac: control well; Ab: zero-adjustment well The experimental results are as Figure 6 shown. Compared with the Con group, the cells in the Model group showed significant damage ( P <0.01). Compared with the Model group, when the concentrations of PSC-2-1 were 50 μM and 100 μM, the cell viability could be significantly improved ( P <0.01); when the concentration of PSC-2-3 reached 100 μM, the cell survival rate could be significantly improved ( P <0.01); within the experimental concentration range (10 - 100 μM), PSC-2-4 did not show a significant regulatory effect on the viability of HK-2 cells ( P >0.05), and PSC-2-H polypeptide at 50 μg / mL could significantly increase the cell survival rate ( P <0.05). There was no statistical significance between the survival rate of the cells in the DMSO group and that in the Control group, and the influence of DMSO as a solvent on HK-2 cells could be excluded. Compared with the Model group, when the concentration of PSC-2-2 reached 25 μM, the cell survival rate could be significantly increased ( P <0.05).

[0036] 2. Conclusion: In the present invention, the polypeptide component was isolated from the dried body of Nectopsyche bilineata by enzymolysis of the dried body of Nectopsyche bilineata, combined with methods such as salting out, Sephadex gel chromatography, and reverse-phase preparative liquid chromatography. The amino acid sequences of the peptide segments in the polypeptide component were identified by LC-MS / MS. The active ingredients in the polypeptide were screened by computer simulation, and their activities were predicted by molecular docking. The renal protective effect of the polypeptide of Nectopsyche bilineata was verified by in vitro cell activity.

[0037] Those skilled in the art can further use the polypeptide of Nectopsyche bilineata of the present invention for the application of preparing drugs with renal protective effects. And further use the polypeptide of Nectopsyche bilineata of the present invention for the application of preparing health products with renal protective effects.

[0038] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, in any aspect, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to include all changes falling within the meaning and scope of the equivalent elements of the claims in the present invention.

[0039] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. The Polypeptide of Nectopsyche, characterized by: The amino acid sequences are as follows: KLGAAPFHFW, IFFLFII, LIRF, and LEI.

2. The extraction process of the dobsonfly polypeptide according to claim 1, wherein It includes the following steps: (1) Using dried Eupolyphaga sinensis Walker as the raw material, extracting polypeptides by enzymatic hydrolysis method, and taking the enzymatic hydrolysate supernatant after centrifugation; (2) Adding alginic acid to the enzymatic hydrolysate supernatant for adsorption and then eluting to obtain a polypeptide solution. After adjusting the pH to the isoelectric point, further separating by salting-out method to obtain the salting-out crude polypeptide; (3) Further separating the salting-out crude polypeptide by Sephadex G-50 dextran gel, detecting the absorbance at 280 nm with an ultraviolet detector, preparing an elution curve, combining the elution peaks and freeze-drying to obtain the crude polypeptide; (4) Using Tris-tricine-SDS-PAGE electrophoresis to detect whether there is polypeptide in the eluted fraction, and subjecting the fraction containing the crude polypeptide to reverse-phase preparative purification to remove small molecule impurities in the Eupolyphaga sinensis Walker polypeptide, intercepting the peak segment and freeze-drying to obtain the Eupolyphaga sinensis Walker polypeptide.

3. The extraction process of the dragonfly larvae polypeptide according to claim 2, characterized in that, In the step (1), after the Eupolyphaga sinensis Walker raw material is crushed, it is defatted by petroleum ether using a Soxhlet extractor, and then the subsequent extraction is carried out.

4. The extraction process of the poly peptide of the dobsonfly larva as described in claim 2, characterized in that, In the step (1), taking Eupolyphaga sinensis Walker powder in a conical flask, adding distilled water to adjust the substrate concentration to 30 mg / mL, adding 1 mol / L HCl to adjust the pH of the solution to 2.0, adding pepsin, hydrolyzing at 37 °C for 1 h, performing a water bath at 80 °C for 10 min to inactivate the protease, centrifuging at 3500 rpm for 15 min to obtain the enzymatic hydrolysate supernatant.

5. The extraction process of the dobsonfly polypeptide according to claim 2, characterized in that, In the step (2), After adding alginic acid to the enzymatic hydrolysate supernatant, stirring for 30 min, then placing it in a refrigerator at 4 °C for 30 min, filtering to obtain alginic acid, eluting with 0.2 mol / L HCl to obtain a polypeptide solution; after adjusting the pH to 3.5 with sodium acetate, adding sodium chloride to a 10% saturation state, salting out overnight in a refrigerator at 4 °C, centrifuging to collect the precipitate, and freeze-drying to obtain the salting-out crude polypeptide.

6. The extraction process of the dobsonfly polypeptide according to claim 5, characterized in that, The 10% saturation state means adding 3.2 g of sodium chloride to every 100 mL of the enzymatic solution.

7. The extraction process of the dobsonfly polypeptide according to claim 2, characterized in that, After swelling Sephadex G-50, it is packed into a chromatography column. The salting-out crude polypeptide is dissolved in deionized water, filtered through a 0.45 μm filter membrane, the sample is slowly added into the chromatography column, and eluted with deionized water; collected regularly with an automatic collector and marked with a receiving tube.

8. The extraction process of the dobsonfly polypeptide according to claim 2, characterized in that, The chromatographic conditions for the reverse-phase preparation are: JADE-PAK ODS AQ C18 chromatographic column, 250 × 10 mm, 5 μm, the sample preparation concentration is 40 mg / mL, the flow rate is 2 mL / min, and the injection volume is 50 μL.

9. The application of the dobsonfly polypeptide according to claim 1, characterized in that, The polypeptide is used for preparing a drug with a renal protection effect.

10. The application of the dobsonfly polypeptide according to claim 1, characterized in that, The polypeptide is used for preparing a health product with a renal protection effect.

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