An anticoagulant earthworm polypeptide and its preparation method and application

Abstract

The present invention belongs to the technical field of polypeptide preparation and application, and specifically relates to an anticoagulant earthworm polypeptide and its preparation method and application. The amino acid sequence of the earthworm polypeptide is IISNFQAR; the earthworm polypeptide preparation method adopts the following steps: (1) crushing and sieving the earthworm, adding water, heating and boiling; (2) cooling the mixed solution obtained in step (1), adjusting the pH value, enzymolysis, boiling, and separation and purification; (3) treating the purified sample obtained in step (2) and performing LC-TOF-MS analysis to obtain the active peptide IISNFQAR. The research on earthworm polypeptide in the present invention confirms its medicinal value for preparing drugs for preventing and treating thrombotic diseases, provides a solid foundation for the clinical application of earthworm polypeptide to treat thrombosis, and provides data support for the development of active peptide substances in animal medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide preparation and application, and specifically relates to an anti-coagulant earthworm polypeptide and a preparation method and application thereof. Background Art

[0002] Modern pharmacological studies have shown that the main role of animal-based traditional Chinese medicine is played by small peptide components. These small peptide components have unique biological activity and low molecular weight. They can show significant therapeutic effects in the treatment of various diseases through multiple mechanisms such as regulating cell signaling pathways, affecting enzyme activity, and immune regulation. With the continuous deepening of scientific research, the pharmacological effects and mechanisms of small peptide components have gradually become clear, providing new ideas and directions for the modernization of animal-based traditional Chinese medicine research and clinical application.

[0003] Pheretima is the dried body of Pheretima seu Radix, Pheretima vulgaris, Pheretima Williamii or Pheretima seu Radix, which belong to the family of the giant earthworms. It is one of the traditional Chinese medicines in my country and has the effects of anticoagulation, promoting fibrinolysis, and improving blood circulation. Clinically, the extract of Pheretima seu Radix has a good anticoagulant effect in vitro and inhibits thrombosis. It is mainly used to prevent and treat cerebral ischemic diseases, cardiovascular diseases, etc. Although previous studies have shown the good anticoagulant effect of Pheretima seu Radix, the research on the molecular size and structure of its anticoagulant active substances is not in-depth enough. Therefore, in-depth research on the effective components of Pheretima seu Radix will not only help to reveal the molecular mechanism of Pheretima seu Radix, but also provide a scientific basis for the development of new anticoagulant drugs. Summary of the invention

[0004] In order to overcome the defects of the prior art, the present invention provides an anticoagulant earthworm polypeptide and a preparation method and application thereof, wherein the earthworm polypeptide exhibits strong anticoagulant activity.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] An anticoagulant earthworm polypeptide, the amino acid sequence of the earthworm polypeptide is IISNFQAR, as shown in SEQ ID No.1; the molecular structure of the earthworm polypeptide is:

[0007] .

[0008] The above earthworm polypeptide is prepared by the following steps:

[0009] (1) Crush and sieve the earthworms, add water, and heat to boil;

[0010] (2) cooling the mixed solution obtained in step (1), adjusting the pH value, performing enzymatic hydrolysis, boiling, and then separating and purifying;

[0011] (3) The purified sample obtained in step (2) was processed and then analyzed by LC-TOF-MS, and the active peptide structure was obtained as IISNFQAR.

[0012] Furthermore, in step (2), the enzymatic hydrolysis method is as follows: after the mixed solution is heated and boiled, it is cooled to about 35-45°C, the pH is adjusted to 1.0-3.0, 0.5-2% pepsin is added, and enzymatic hydrolysis is carried out at 30-40°C for 1-2 h, the pH is adjusted to 7.0-9.0, 0.5-2% trypsin is added, and the mixture is kept warm for 3-5 h, and boiled for 10-15 min; the separation and purification is centrifuged at 3500-4500 rpm for 15-20 min, the supernatant is ultrafiltered through a 3 KD ultrafiltration membrane, desalted by electrodialysis or dialysis bag, and then subjected to cationic resin and RP-HPLC semi-preparative separation and purification.

[0013] Furthermore, the nano-liquid chromatography column conditions for the LC-TOF-MS analysis in step (3) are as follows: C18 reverse phase analytical column 75 mm×20 cm×3 µm; mobile phase A: a mixture of 99.9% water and 0.1% formic acid, mobile phase B: a mixture of 80% acetonitrile and 0.1% formic acid; liquid phase gradient elution is: 0-2 min, 2-6% B; 2-95 min, 6-20% B; 95-107 min, 20-32% B; 107-108 min, 32-100% B; 108-120 min, 100% B; and the mobile phase flow rate is 300 nL / min.

[0014] Furthermore, in step (3), the method for treating the purified sample obtained in step (2) is as follows: 1 to 5 mg of the purified sample in step (2) is added to 200 µL of 25 mM ammonium bicarbonate solution to dissolve, 2 µL of 1M DTT is added and placed in a 60°C water bath for >40 min, and then 8 µL of freshly prepared 1 M iodoacetamide is added and placed at room temperature away from light for 30 min; desalting treatment is performed, and the dried sample is added to 10 µL of loading buffer and oscillated to dissolve.

[0015] Furthermore, in step (1), the standard mesh size of the earthworm crushed and sieved is 80 mesh; the weight of the added water is 8 to 12 times the weight of the earthworm; and the heating and boiling time is 15 to 30 minutes.

[0016] An application of the above earthworm polypeptide, wherein the earthworm polypeptide is used for preparing a medicine for preventing and treating thrombotic diseases.

[0017] Furthermore, the polypeptide or its derivative or composition is used as an active ingredient directly or in combination with a pharmaceutical carrier to prepare a preparation.

[0018] Furthermore, the preparation is an injection, tablet, capsule, aerosol or drop.

[0019] Beneficial effects: (1) The polypeptide structure described in the present invention is a high-content, high-activity small molecule bioactive peptide component obtained by refining and purifying small molecule peptides of earthworm biomimetic enzymatic hydrolysis and analyzing. This small molecule bioactive peptide component is discovered for the first time and has not been reported.

[0020] (2) The research on the earthworm polypeptide IISNFQAR in the present invention confirms the medicinal value of preventing and treating thrombotic diseases, provides a solid foundation for the clinical application of the earthworm polypeptide IISNFQAR in treating thrombosis, and provides data support for the development of active peptide substances for animal medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their description are used to explain the present invention, and do not constitute an improper limitation of the present invention.

[0022] Figure 1 This is a non-continuous intermittent constant volume operation diagram;

[0023] Figure 2 This is the separation effect diagram of Sephadex G15 in the 75% ethanol elution area. DETAILED DESCRIPTION

[0024] The present invention is further illustrated by specific examples below. It should be understood that the preparation methods in the examples of the present invention are only used to illustrate the present invention, rather than to limit the present invention. Under the premise of the concept of the present invention, simple improvements to the preparation methods of the present invention fall within the scope of protection required by the present invention.

[0025] It should also be noted that the various preferred technical features of the method of the present invention mentioned above and the various specific technical features in the embodiments described in detail below can be combined together, and all numerical ranges with the numerical values ​​specifically disclosed in the present invention as upper and lower limits and the like of various combinations of all these technical features fall within the scope of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources or synthesized from raw materials obtained from commercial sources.

[0028] The specific embodiments of the present invention are described in detail below in conjunction with the technical solutions, but the process conditions are not limited to these embodiments.

[0029] The present invention will be further described below in conjunction with specific embodiments:

[0030] Example 1 Preparation of Pheretima Polypeptide

[0031] (1) Preparation of earthworm biomimetic enzymatic hydrolysate

[0032] ① Crush 200 g of earthworms through an 80-mesh sieve, add 10 times the weight of water, heat and boil for 25 min, cool to about 40°C, adjust the pH to 2.0, add 0.5-2% pepsin, enzymatically hydrolyze at 40°C for 1 h, adjust the pH to 8.0, add 1.5% trypsin, keep warm for 4 h, boil for 12 min, centrifuge at 4000 rpm for 18 min, and take the supernatant to obtain the earthworm bionic enzymatic extract;

[0033] ② Take the earthworm biomimetic enzymatic hydrolysate, dilute it 1-fold and adjust the pH to 7.0, pour it into the sample tank of the electrodialysis instrument, add tap water and deionized water to the electrode pool and desalination pool respectively. Adjust the voltage to 20 V and control the flow rate to 10 L·h -1 , taking the change of the conductivity value of the enzymatic hydrolysate in the sample tank as the detection index, stopping the electrodialysis desalination operation after the conductivity value approaches stability, and collecting the desalted enzymatic hydrolysate;

[0034] ③ Take the desalted enzymatic hydrolysate prepared in step ② and use a 3000 Da ultrafiltration membrane at 0.8 MPa using a discontinuous intermittent constant volume operation (see Figure 1 ), the unmembrane enzymatic hydrolysate is a polypeptide solution of >3000 Da, the membrane-passed enzymatic hydrolysate is a polypeptide solution of <3000 Da, the polypeptide solution of <3000 Da is taken and separated by a 1000 Da nanofiltration membrane at 0.8 MPa using a discontinuous intermittent constant volume operation to obtain a polypeptide solution of 1000-3000 Da that has not passed through the membrane and a polypeptide solution of <1000 Da that has passed through the membrane, the above polypeptide solutions are respectively concentrated under reduced pressure and freeze-dried to obtain polypeptide freeze-dried powders of <1000 Da, 1000-3000 Da, and >3000 Da for standby use;

[0035] ④ Take the <1000Da peptide prepared in step ③ and distilled water to make 20 mg·mL -1 , continuous loading, loading flow rate is 2 BV·h -1 , A 220nm ≥0.10 is the loading end point, the effluent is collected, static adsorption is performed for 3 hours, and then eluted with water to A 220nm ≤0.05, the eluent and the effluent were combined as water eluent, and washed with 25% ethanol, 50% ethanol, and 75% ethanol respectively until A 220nm ≤0.05, collect the eluates, concentrate under reduced pressure, freeze-dry and set aside;

[0036] ⑤ Take an appropriate amount of Sephadex G15 gel, activate it, and load it into a column (1.6×75 cm). Prepare the lyophilized powder of the 75% ethanol elution fraction into 8 mg·mL -1 0.02 BV was loaded after filtering with 0.45 μm membrane, and eluted with deionized water. The flow rate was controlled at 2.0 mL min -1 1 test tube was collected for every 2.0 mL, and each test tube was numbered according to the order of collection and the absorbance was measured at 280 nm and 220 nm under ultraviolet light. The test tube number and the absorbance change value were plotted. The results are shown in Figure 2 .Depend on Figure 2 It can be seen that the samples showed three main peaks after Sephadex G15 separation, which were named F1, F2 and F3. The same peaks were combined, concentrated under reduced pressure, freeze-dried and used for later use;

[0037] ⑥ Prepare the chromatographic peak samples in step ⑤ with deionized water to 20 mg·mL -1 The Fib-TT and polypeptide contents of each peak were determined. The results are shown in Table 1.

[0038] Table 1 Anticoagulant activity and peptide content determination results of each sample

[0039]

[0040] (2) LC-TOF-MS separation

[0041] Take 1 g of the purified F2 sample in step (1) and add 200 µL of 25 mM ammonium bicarbonate solution to dissolve it. Add 2 µL of 1 M DTT and place it in a 60°C water bath for >40 min. Then add 8 µL of freshly prepared 1 M iodoacetamide and place it at room temperature in the dark for 30 min. Desalt the sample using a micro desalting column. Add 10 µL of loading buffer to the dried sample and shake it to dissolve it. Determine the composition using the following method:

[0042] Nano-liquid chromatography column: C18 reverse phase column (75 mm×20 cm×3 µm); mobile phase A: 99.9% water and 0.1% formic acid mixture, mobile phase B: 80% acetonitrile and 0.1% formic acid mixture. Liquid phase gradient elution method: 0-2 min, 2-6% B; 2-95 min, 6-20% B; 95-107 min, 20%-32% B; 107-108 min, 32-100% B; 108-120 min, 100% B. The mobile phase flow rate was 300 nL / min. The full scan acquisition (m / z 350-1800) was performed in ESI+ mode with data-dependent scanning mode in the orbitrap at a resolution of 70,000 (AGC 3e6), a normalized collision energy (NCE) of 28.0, a capillary temperature of 275 °C, a spray voltage of 1800 V, and a MS 2 Resolution 17500 (AGC 1e5), full scan and MS 2 The maximum fill time of the scans was set to 50 ms and 45 ms, respectively, and the dynamic exclusion time was set to 30 s.

[0043] (3) Mass spectrometry data analysis

[0044] The Peaks software search engine was used to perform sequence and relative quantitative analysis on the data of the test products. The analysis parameters were: primary spectrum mass tolerance: 10 ppm, secondary spectrum mass tolerance: 0.020 Da, variable modifications: Deamidation (NQ), Oxidation (M), non-enzymatic cleavage, and the false positive rate FDR of peptides and proteins was less than 1%. The Peptidemass function provided by Uniprot simulated the results of pepsin and trypsin digestion of proteins from different species. The characteristic peptide sequences of earthworms relative to other species were obtained by aligning the protein sequences of earthworms with those of other species. The mass spectrometry data were searched using ProteomeDiscoverer software (version 2.2). According to the principles of (1) 6-10 amino acids, (2) avoiding peptides that are prone to artificial modification as much as possible, and (3) no missed cleavage sites during enzyme cleavage, combined with mass spectrometry response and separation, "VALSVIQR", "AVFPSLVGR", "VIPSLNGK", "IISNFQAR", and "FEEILPIYK" were determined as characteristic peptides of earthworms. IISNFQAR is characteristic polypeptide 1, as shown in SEQ ID No. 1; VALSVIQR is characteristic polypeptide 2, as shown in SEQ ID No. 2; AVFPSLVGR is characteristic polypeptide 3, as shown in SEQ ID No. 3; VIPSLNGK is characteristic polypeptide 4, as shown in SEQ ID No. 4; FEEILPIYK is characteristic polypeptide 5, as shown in SEQ ID No. 5. Since this sample was prepared by bionic enzymatic hydrolysis, pepsin and trypsin were introduced in the preparation process, it is necessary to identify the source of the polypeptide sequence.

[0045] (4) Identification of peptide sources

[0046] The pepsin and trypsin used in this study were both derived from pigs. A protease search was performed using the UniProt (https: / / www.uniprot.org / ) database. Comparison of VALSVIQR, AVFPSLVGR, VIPSLNGK, IISNFQAR, and FEEILPIYK showed no intersection with pig pepsin and trypsin, suggesting that the polypeptide sequence was not derived from pig pepsin and trypsin. Therefore, we concluded that the five polypeptide sequences were all derived from earthworms.

[0047] (5) Amino acid sequence analysis and structure prediction

[0048] Using PepDraw (http: / / www.pepdraw.com / ) and PEP-FOLD3

[0049] The molecular structures and configurations of the five peptide chains were predicted using the database (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD3 / ), and the results are shown in Table 2.

[0050] Table 2 Results of physicochemical property analysis of peptide sequences

[0051]

[0052] The structure diagram and configuration prediction of the characteristic peptide IISNFQAR are:

[0053] ;

[0054] The structure diagram and configuration prediction of the characteristic peptide VALSVIQR are:

[0055] ;

[0056] The structural diagram and configuration of the characteristic polypeptide AVFPSLVGR are as follows:

[0057] ;

[0058] The structural diagram and configuration of the characteristic polypeptide VIPSLNGK are as follows:

[0059] ;

[0060] The structural diagram and configuration of the characteristic polypeptide FEEILPIYK are:

[0061] .

[0062] Example 2 Study on the anticoagulant activity of earthworm polypeptide in vitro

[0063] The five peptide sequences of VALSVIQR, AVFPSLVGR, VIPSLNGK, IISNFQAR, and FEEILPIYK were commissioned to Shanghai Jier Biochemical Co., Ltd. for solid phase synthesis. The purity was >95%. The synthetic peptides were added with deionized water to prepare 20 mg mL -1 The anticoagulant activity was determined by the Fibg-TT method according to the kit instructions. The results are shown in Table 3.

[0064] Table 3 Synthetic polypeptide anticoagulant activity test results

[0065] Serial number Sample (20 mg / mL) Fib-TT(s) 1 Water Blank 16.52 2 VALSVIQR 19.20 3 AVFPSLVGR 17.60 4 VIPSLNGK 20.96 5 IISNFQAR 62.32 6 FEEILPIYK 24.72

[0066] This study conducted an in-depth analysis of the top 5 polypeptide sequences. By comparing with the amino acid sequences of pepsin and trypsin, the possibility of foreign matter introducing into the polypeptides was ruled out. The molecular structure and configuration of the 5 polypeptides were predicted through the database, and solid phase synthesis and anticoagulant activity were performed on them. The results showed that only the polypeptide IISNFQAR showed strong anticoagulant activity and was the key component in earthworms to exert anticoagulant activity.

[0067] The amino acid sequence of porcine pepsin is shown in SEO D No.6, and the amino acid sequence of porcine trypsin is shown in SEO D No.7.

[0068] Example 3 Study on the anticoagulant activity of earthworm polypeptide in vivo

[0069] 1 Experimental content

[0070] (1) Establishment of rat model of cold stagnation and blood stasis

[0071] After 7 days of adaptive feeding, 64 SD rats were randomly divided into 8 groups, namely, blank group, model group, positive control group (aspirin group), VALSVIQR group, AVFPSLVGR group, VIPSLNGK group, IISNFQAR group and FEEILPIYK group, with 8 rats in each group. Except for the blank group, rats in the other groups were subcutaneously injected with epinephrine hydrochloride (0.9 mg / kg) twice, with an interval of 4 hours. Two hours after the first subcutaneous injection, the rats were placed in ice water (0-4℃) for 5 minutes. The above operation was repeated for 15 days to replicate the acute blood stasis model.

[0072] (2) Animal dosing regimen

[0073] Starting from the 8th day of modeling, the drug group was gavaged 1 hour after the second injection of epinephrine hydrochloride for 7 consecutive days. The dosage was set according to the clinical dosage of humans. The dosage of the positive control group was 0.2 g / kg (the solvent was normal saline), and the dosage of the peptide was gavaged at 10 mg / kg. At the same time, the blank group and the model group rats were gavaged with the same volume of normal saline in the same time period, once a day, for 8 consecutive days.

[0074] (3) Blood sample collection

[0075] After the last administration, the rats in each group were fasted but not watered for 12 h.

[0076] Blood was collected from the artery, part of which was packed into heparin anticoagulant tubes, and the rest of the whole blood was added into anticoagulant tubes containing 3.2% sodium citrate (the volume ratio of blood to anticoagulant was 9:1).

[0077] (4) Detection of blood rheology related indicators

[0078] Turn on the blood rheometer and preheat for 15 min. Take an appropriate amount of whole blood from the heparin anticoagulation tube in step (3) and measure the blood rheology at low, medium and high shear rates (10 S -1 , 50 S -1 , 200 S -1 The remaining whole blood was centrifuged at 3500 rpm for 15 min at 4 °C to measure the plasma viscosity.

[0079] (5) Detection of coagulation system related indicators

[0080] Take the whole blood anticoagulated with 3.2% sodium citrate in step (3) and centrifuge at 3500 rpm for 15 min at 4°C to prepare whole blood. Use an automatic coagulometer to measure prothrombin time (PT), activated partial prothrombin time (APTT) and thrombin time (TT). The operation is carried out according to the instrument instructions.

[0081] (6) Data processing

[0082] GraphPad Prism 5.0 software was used to analyze the data, and the experimental results were presented as mean ± standard deviation (x ± s). Two-way t-test or two-way analysis of variance was used to test the differences between the groups. p When <0.05, the difference was statistically significant.

[0083] 2 Experimental Results

[0084] (1) Effects of leech active peptides on blood rheology in rats with cold-induced blood stasis

[0085] Blood viscosity and plasma viscosity reflect macroscopic blood rheology, which can more intuitively reflect the effect of drugs on blood properties. The results of the effects of peptides on whole blood and plasma viscosity are shown in Table 4. Compared with the blank group, the whole blood viscosity and plasma viscosity of the model group at different shear rates increased significantly, indicating that the model was successfully established. Compared with the model group, the positive control group (aspirin group) can significantly reduce the whole blood viscosity and plasma viscosity at different shear rates, the IISNFQAR group can significantly reduce the whole blood viscosity and plasma viscosity at high, medium and low shear rates, and the VALSVIQR group, AVFPSLVGR group, VIPSLNGK group and FEEILPIYK group have no significant difference compared with the model group, but there is also a trend of reducing the medium and low shear viscosity of whole blood and plasma viscosity, which also indicates that it has a certain efficacy.

[0086] Table 4 Effects of peptides on whole blood and plasma viscosity in rats with cold coagulation and blood stasis model (n=8)

[0087]

[0088] Note: Compared with the blank group,## p <0.01; compared with the model group, * p <0.05,** p <0.01.

[0089] (2) Effects of leech active peptides on coagulation parameters in rats with cold-induced blood stasis

[0090] The four coagulation tests are the basic indicators for detecting the coagulation function of the human body. Among them, PT and APTT reflect the exogenous pathway and the intrinsic pathway of blood coagulation, respectively. As shown in Table 5, compared with the blank group, the PT, TT and APTT of the model group rats were significantly reduced, indicating that the model was successfully established. Compared with the model group, the positive control group can significantly prolong the PT, TT and APTT time, the IISNFQAR group can significantly prolong the PT, TT and APTT time, and the VALSVIQR group, AVFPSLVGR group, VIPSLNGK group and FEEILPIYK group have a certain prolongation effect on the PT time, but it is not significant.

[0091] The above results show that IISNFQAR has a good anticoagulant effect.

[0092] Table 5 Effects of peptides on coagulation indexes in rats with cold-coagulation and blood stasis model (n=8)

[0093]

[0094] Note: Compared with the blank group, ## p <0.01; compared with the model group, * p <0.05,** p <0.01

[0095] Although the above describes the specific implementation mode of the invention in conjunction with the drawings, it is not intended to limit the scope of protection of the invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. An anticoagulant earthworm polypeptide, characterized in that: The amino acid sequence of the earthworm polypeptide is IISNFQAR; the molecular structure of the earthworm polypeptide is: 。 2. An application of the anticoagulant earthworm polypeptide according to claim 1, characterized in that: The earthworm polypeptide is used for preparing anticoagulant drugs.

3. The use according to claim 2, characterized in that: The earthworm polypeptide is used as an active ingredient and combined with a pharmaceutical carrier to prepare a preparation.

4. The use according to claim 3, characterized in that: The preparation is injection, tablet, capsule, aerosol or drop.

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