A camel milk casein anticoagulant peptide, preparation method and application

Abstract

The application provides a camel milk casein anticoagulant peptide, a preparation method and application, and the camel milk casein anticoagulant peptide is ISQFYQKWK. The application takes camel milk casein as raw material, obtains the target anticoagulant peptide through enzymolysis, separation and identification and solid-phase synthesis, solves the problems that existing anticoagulant drugs have bleeding risk, are relatively high in cost and are limited in long-term application, and simultaneously improves the high-value utilization level of camel milk casein resources. The camel milk casein anticoagulant peptide is natural in origin, clear in sequence, has good in-vitro anticoagulant activity, can intervene in the blood clotting process by prolonging the activated partial thromboplastin time, the prothrombin time and the thrombin time.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a camel milk casein anticoagulant peptide, its preparation method, and its application. Background Technology

[0002] Thrombosis refers to the abnormal adhesion and aggregation of formed elements in the blood within the lumen of blood vessels, gradually forming a clot. Its main components include fibrin, platelets, and embedded red blood cells and white blood cells. It is usually closely related to factors such as vascular endothelial damage, abnormal blood flow, and hypercoagulability. Once a thrombus forms, it can cause narrowing or blockage of blood vessels, affecting the blood supply to local tissues and organs. In severe cases, it can lead to diseases such as myocardial infarction, cerebral infarction, and deep vein thrombosis. Due to the insidious onset, rapid progression, and high mortality and disability rates associated with thrombotic diseases, it has become a significant public health problem threatening human health.

[0003] Currently, anticoagulants used clinically for the prevention and treatment of thrombotic diseases mainly include heparin, hirudin, and their derivatives. While these drugs can effectively delay thrombus formation by intervening in the coagulation cascade, their practical application still has certain limitations, such as high treatment costs, significant bleeding risk, insufficient long-term medication adherence, and significant individual variability in response. Therefore, finding safe, well-defined, and suitable natural anticoagulant active ingredients for further development and utilization has become an important direction in antithrombotic research.

[0004] Milk protein is a class of natural protein resources with high nutritional value and a relatively well-defined structure. During enzymatic hydrolysis, fermentation, or digestion, it can release a variety of bioactive peptides with physiological regulatory functions. Existing research has shown that milk-derived bioactive peptides have certain functional potential in antioxidation, blood pressure reduction, immune regulation, antibacterial activity, and anticoagulation. Among them, milk-derived anticoagulant peptides can intervene in thrombosis by inhibiting thrombin activity, delaying fibrinogen production, or regulating platelet-related processes, showing promising development prospects. Camel milk, as a unique milk resource, has a distinctive protein composition and contains abundant potentially bioactive peptide precursor sequences. Summary of the Invention

[0005] Based on the above background, this invention aims to provide a camel milk casein anticoagulant peptide, its preparation method, and its application, to solve the following problems existing in the prior art: First, although commonly used anticoagulant drugs have clear efficacy, they still have problems such as bleeding risk, high cost, limited long-term use, and insufficient applicability to some individuals; second, camel milk, as a special milk resource, contains many potential bioactive peptide precursors, but its anticoagulant active components have not been fully explored and utilized; third, the existing research on the preparation, screening, identification, and synthesis verification system of camel milk casein anticoagulant peptides is still imperfect, making it difficult to stably obtain camel milk-derived anticoagulant peptides with well-defined sequences and good activity. Therefore, this invention establishes a technical route of "camel milk casein extraction - enzymatic hydrolysis preparation - peptide identification - solid-phase synthesis - activity verification" to achieve the effective acquisition and application development of camel milk casein anticoagulant peptides.

[0006] To solve the above-mentioned technical problems, one of the objectives of this invention is to provide a camel milk casein anticoagulant peptide, wherein the camel milk casein anticoagulant peptide is ISQFYQKWK; The amino acid sequence of the camel milk casein anticoagulant peptide ISQFYQKWK is SEQ ID NO: 1.

[0007] Based on the same inventive concept, the present invention also provides a method for preparing camel milk casein anticoagulant peptides, comprising the following steps: S1: Centrifuge raw camel milk to remove the upper fat layer, take the lower clear liquid and adjust the pH to 4.6, centrifuge to collect the precipitate, add -20℃ pre-cooled n-hexane, stir at 4℃ for 24 hours to defatting, and change the liquid several times. After filtering through a Buchner funnel, freeze dry for 24 hours to obtain casein. S2: Dissolve casein in Tris-HCl buffer at pH 8.0 to prepare a casein solution with a concentration of 40 mg / mL. Add neutral protease for enzymatic hydrolysis to obtain the hydrolysate. S3: Dilute the enzymatic hydrolysate to 1-5 mg / mL, acidify with formic acid to pH < 3.0, treat with an Oasis HLB solid-phase extraction desalting column, collect the effluent, concentrate by vacuum centrifugation, and dry to obtain crude polypeptide containing several polypeptides. S4: The peptide was identified using ion mobility high-resolution mass spectrometry, and camel milk casein anticoagulant peptide ISQFYQKWK with high anticoagulant activity was obtained.

[0008] Preferably, in S1, the first centrifugation condition is 7500×g and the centrifugation time is 20 min; the second centrifugation condition is 9000×g and the centrifugation time is 20 min; the solid-liquid ratio of the precipitate to n-hexane is 1:10; in S3, the centrifugation condition is 500×g.

[0009] Preferably, in step S2, the enzyme dosage is 2500 U / g, the enzymatic hydrolysis temperature is 40℃, and the enzymatic hydrolysis time is 30 min.

[0010] Preferably, in step S3, the Oasis HLB solid-phase extraction desalting column treatment and crude peptide collection steps include: S31: Add 1 mL of methanol to the solid phase extraction desalting column and let it flow down by gravity, then discard the effluent; S32: Add 700 µL Buffer B, pressurize and elute the column, repeat twice, and discard the eluent; S33: Add 700 µL Buffer A, pressurize and elute from the column, then discard the eluent; S34: Dilute the enzyme digest sample to twice its volume with Buffer A, load 700 µL of the sample onto the column, allow it to flow down by gravity, and collect the eluent.

[0011] This invention also provides a solid-phase synthesis method for camel milk casein anticoagulant peptides, comprising the following steps: S1: Soak 2-chlorotriphenylmethyl chloride resin in dichloromethane to make it fully swollen. After removing the dichloromethane from the surface of the 2-chlorotriphenylmethyl chloride resin, clean the 2-chlorotriphenylmethyl chloride resin with N,N-dimethylformamide. S2: Using the 2-chlorotriphenylmethyl chloride resin treated with S1 as a solid support, Fmoc-amino acids were dissolved in N,N-dimethylformamide, and diisopropylethylamine was added to react fully to achieve the initial connection between amino acids and resin. S3: Add a mixed solution of methanol, diisopropylethylamine and dichloromethane, and react at room temperature for 0.5 h to block unreacted active sites on the resin; S4: Add a mixed solution of piperidine and N,N-dimethylformamide, react for 0.5 h to remove the Fmoc protecting group; S5: Dissolve Fmoc-amino acids and benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate in N,N-dimethylformamide, add diisopropylethylamine, and react at room temperature for 2-4 h to complete the coupling. Repeat S4 in sequence, and then carry out the next round of coupling until all amino acids are linked according to the target sequence. S6: Add a cleaving agent to the coupled 2-chlorotriphenylmethyl chloride resin, react at room temperature for 2 h, filter the reaction solution to remove resin, collect the filtrate and remove impurities by rotary evaporation, add excess ice-cold ether to precipitate, centrifuge to collect the precipitate, wash twice with ice-cold ether and then vacuum dry to obtain crude camel milk casein anticoagulant peptide ISQFYQKWK. S7: The crude camel milk casein anticoagulant peptide was detected by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry to confirm the presence of the target peptide. If the target molecular weight peak was detected, the crude camel milk casein anticoagulant peptide was purified by semi-preparative high performance liquid chromatography using an aqueous solution containing 0.1% trifluoroacetic acid and an acetonitrile solution containing 0.1% trifluoroacetic acid as the mobile phase. S8: Collect the target peak components, concentrate them by rotary evaporation, and then perform matrix-assisted laser desorption / ionization time-of-flight mass spectrometry to confirm the components. Finally, use analytical HPLC to determine the purity.

[0012] Preferably, the amino acids used in preparing the camel milk casein anticoagulant peptide ISQFYQKWK are: Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Trp(Boc)-OH.

[0013] Preferably, in step S3, the volume ratio of methanol, diisopropylethylamine, and dichloromethane is 2:1:17; and in step S4, the volume ratio of piperidine and N,N-dimethylformamide is 1:4.

[0014] Preferably, in step S6, the cutting agent is a mixture of trifluoroacetic acid, triisobutylsilane, and deionized water, wherein the volume ratio of trifluoroacetic acid, triisobutylsilane, and deionized water is 95:2.5:2.5.

[0015] The present invention also provides an application of camel milk casein anticoagulant peptide, which is used to prepare a pharmaceutical preparation with antithrombotic function.

[0016] The above-described one or more technical solutions of the present invention have the following technical effects: 1. Naturally derived with good application safety: The anticoagulant peptides described in this invention are derived from camel milk casein, a raw material with natural properties and a good edible basis. Compared with traditional anticoagulant drugs such as heparin and its derivatives, hirudin and its analogues, these milk-derived polypeptides have the advantages of fewer potential side effects and suitability for further development into oral functional ingredients. They can, to some extent, compensate for the shortcomings of existing anticoagulant drugs, such as high price, limited long-term use, bleeding risk, and individual adaptability differences, providing a new option for the development of naturally derived anticoagulant active substances.

[0017] 2. Enhancing the High-Value Utilization of Camel Milk Casein: This invention uses camel milk casein as raw material and, through enzymatic hydrolysis, separation and screening, and mass spectrometry identification, obtains specific peptides with anticoagulant activity from camel milk protein resources, achieving effective release and precise screening of potential active peptide precursor sequences. This method helps expand the application of camel milk resources in functional foods, health products, and pharmaceutical preparations, and increases the added value of deep-processed camel milk products.

[0018] 3. Complete preparation route and strong process reproducibility: This invention establishes a systematic technical process including camel milk casein extraction, enzymatic hydrolysis preparation, peptide separation and identification, solid-phase synthesis, and activity verification. The key conditions in this preparation method are clearly defined; for example, the enzymatic hydrolysis parameters, solid-phase synthesis steps, and purification and detection methods all possess strong operability and reproducibility, providing a technical foundation for the stable preparation, batch acquisition, and subsequent application research of camel milk casein anticoagulant peptides.

[0019] 4. Clear and efficient anticoagulant activity with a comprehensive mechanism of action: Experimental verification shows that camel milk casein anticoagulant peptide ISQFYQKWK exerts its anticoagulant effect by influencing the intrinsic coagulation pathway (prolonging activated partial thromboplastin time), the extrinsic coagulation pathway (prolonging prothrombin time), and fibrinogen conversion (prolonging thrombin time). The anticoagulant activity of camel milk casein anticoagulant peptide ISQFYQKWK is relatively high, with an anticoagulant activity unit of 98.87 ATU / mg, indicating that camel milk casein can release active peptides with good anticoagulant potential.

[0020] 5. Diverse Applications and Promising Development Prospects: The camel milk casein anticoagulant peptides described in this invention possess definite in vitro anticoagulant activity and can be further used as functional ingredients in the development of antithrombotic products. Its applications can cover functional dairy products, deep-processed camel milk products, health foods, and pharmaceutical preparations, helping to expand the utilization pathways of camel milk resources, enrich the product types of naturally derived anticoagulant active ingredients, and provide a new technological foundation for the research and development of related functional products. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0022] Figure 1 Mass spectrometry analysis of camel milk casein anticoagulant peptide ISQFYQKWK; Figure 2 Camel milk casein anticoagulant peptide ISQFYQKWK high performance liquid chromatography analysis diagram; Figure 3 Graph showing the anticoagulant properties of enzymatic hydrolysate of camel milk casein; Figure 4A graph showing the anticoagulant activity of camel milk casein anticoagulant peptide ISQFYQKWK. Detailed Implementation

[0023] This invention provides a camel milk casein anticoagulant peptide, wherein the camel milk casein anticoagulant peptide is ISQFYQKWK; The amino acid sequence of the camel milk casein anticoagulant peptide ISQFYQKWK is SEQ ID NO: 1.

[0024] This invention also provides a method for preparing camel milk casein anticoagulant peptides, comprising the following steps: S1: Centrifuge raw camel milk to remove the upper fat layer, take the lower clear liquid and adjust the pH to 4.6, centrifuge to collect the precipitate, add -20℃ pre-cooled n-hexane, stir at 4℃ for 24 hours to defatting, and change the liquid several times. After filtering through a Buchner funnel, freeze dry for 24 hours to obtain casein. S2: Dissolve casein in Tris-HCl buffer at pH 8.0 to prepare a casein solution with a concentration of 40 mg / mL. Add neutral protease for enzymatic hydrolysis to obtain the hydrolysate. S3: Dilute the enzymatic hydrolysate to 1-5 mg / mL, acidify with formic acid to pH < 3.0, treat with an Oasis HLB solid-phase extraction desalting column, collect the effluent, concentrate by vacuum centrifugation, and dry to obtain crude polypeptide containing several polypeptides. S4: The peptide was identified using ion mobility high-resolution mass spectrometry, and camel milk casein anticoagulant peptide ISQFYQKWK with high anticoagulant activity was obtained.

[0025] Preferably, in S1, the first centrifugation condition is 7500×g and the centrifugation time is 20 min; the second centrifugation condition is 9000×g and the centrifugation time is 20 min; the solid-liquid ratio of the precipitate to n-hexane is 1:10; in S3, the centrifugation condition is 500×g.

[0026] Preferably, in step S2, the enzyme dosage is 2500 U / g, the enzymatic hydrolysis temperature is 40℃, and the enzymatic hydrolysis time is 30 min.

[0027] Preferably, in step S3, the Oasis HLB solid-phase extraction desalting column treatment and crude peptide collection steps include: S31: Add 1 mL of methanol to the solid phase extraction desalting column and let it flow down by gravity, then discard the effluent; S32: Add 700 µL Buffer B, pressurize and elute the column, repeat twice, and discard the eluent; S33: Add 700 µL Buffer A, pressurize and elute from the column, then discard the eluent; S34: Dilute the enzyme digest sample to twice its volume with Buffer A, load 700 µL of the sample onto the column, allow it to flow down by gravity, and collect the eluent.

[0028] This invention also provides a solid-phase synthesis method for camel milk casein anticoagulant peptides, comprising the following steps: S1: Soak 2-chlorotriphenylmethyl chloride resin in dichloromethane to make it fully swollen. After removing the dichloromethane from the surface of the 2-chlorotriphenylmethyl chloride resin, clean the 2-chlorotriphenylmethyl chloride resin with N,N-dimethylformamide. S2: Using the 2-chlorotriphenylmethyl chloride resin treated with S1 as a solid support, Fmoc-amino acids were dissolved in N,N-dimethylformamide, and diisopropylethylamine was added to react fully to achieve the initial connection between amino acids and resin. S3: Add a mixed solution of methanol, diisopropylethylamine and dichloromethane, and react at room temperature for 0.5 h to block unreacted active sites on the resin; S4: Add a mixed solution of piperidine and N,N-dimethylformamide, react for 0.5 h to remove the Fmoc protecting group; S5: Dissolve Fmoc-amino acids and benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate in N,N-dimethylformamide, add diisopropylethylamine, and react at room temperature for 2-4 h to complete the coupling. Repeat S4 in sequence, and then carry out the next round of coupling until all amino acids are linked according to the target sequence. S6: Add a cleaving agent to the coupled 2-chlorotriphenylmethyl chloride resin, react at room temperature for 2 h, filter the reaction solution to remove resin, collect the filtrate and remove impurities by rotary evaporation, add excess ice-cold ether to precipitate, centrifuge to collect the precipitate, wash twice with ice-cold ether and then vacuum dry to obtain crude camel milk casein anticoagulant peptide ISQFYQKWK. S7: The crude camel milk casein anticoagulant peptide was detected by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry to confirm the presence of the target peptide. If the target molecular weight peak was detected, the crude camel milk casein anticoagulant peptide was purified by semi-preparative high performance liquid chromatography using an aqueous solution containing 0.1% trifluoroacetic acid and an acetonitrile solution containing 0.1% trifluoroacetic acid as the mobile phase. S8: Collect the target peak components, concentrate them by rotary evaporation, and then perform matrix-assisted laser desorption / ionization time-of-flight mass spectrometry to confirm the components. Finally, use analytical HPLC to determine the purity.

[0029] Preferably, the amino acids used in preparing the camel milk casein anticoagulant peptide ISQFYQKWK are: Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Trp(Boc)-OH.

[0030] Preferably, in step S3, the volume ratio of methanol, diisopropylethylamine, and dichloromethane is 2:1:17; and in step S4, the volume ratio of piperidine and N,N-dimethylformamide is 1:4.

[0031] Preferably, in step S6, the cutting agent is a mixture of trifluoroacetic acid, triisobutylsilane, and deionized water, wherein the volume ratio of trifluoroacetic acid, triisobutylsilane, and deionized water is 95:2.5:2.5.

[0032] This invention also provides an application of camel milk casein anticoagulant peptide, which is used to prepare a pharmaceutical preparation with antithrombotic function.

[0033] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0034] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0035] Example 1: Preparation of enzymatic hydrolysate S1: Centrifuge raw camel milk at 7500×g for 20 minutes to remove the upper fat layer. Adjust the pH of the lower supernatant to 4.6 using 10% acetic acid. Centrifuge at 9000×g for 20 minutes, collect the precipitate, add pre-cooled n-hexane at -20℃, and the solid-liquid ratio of precipitate to n-hexane is 1:10 (g / mL). Defatt the mixture by stirring at 4℃ for 24 hours, changing the liquid multiple times. Filter through a Buchner funnel and freeze-dry for 24 hours to obtain casein. S2: Dissolve casein in Tris-HCl buffer at pH 8.0 to prepare a casein solution with a concentration of 40 mg / mL. Add neutral protease for enzymatic hydrolysis at an enzyme dosage of 2500 U / g, a hydrolysis temperature of 40℃, and a hydrolysis time of 30 min. After hydrolysis, inactivate the enzyme by boiling in a water bath for 10 min to obtain the hydrolysate.

[0036] Example 2: Isolation and Identification of Camel Milk Casein Anticoagulant Peptides S1: Dilute the enzymatic hydrolysate obtained in Example 1 to 1-5 mg / mL, and then acidify it with formic acid to pH < 3.0; S2: Add 1 mL of methanol to the Oasis HLB solid-phase extraction desalting column and allow it to flow down by gravity. Discard the eluent. Add 700 µL of Buffer B (80% ACN, 0.2% TFA) and allow the column to flow out under pressure. Repeat twice and discard the eluent. Add 700 µL of Buffer A (0.2% TFA) and allow the column to flow out under pressure. Discard the eluent. Dilute the enzyme digest obtained in S1 to twice the volume with Buffer A. Load 700 µL of sample onto the column and allow it to flow down by gravity. Collect the eluent and concentrate it by vacuum centrifugation using an integrated vacuum centrifuge (centrifugation conditions: 500×g) until completely dry to obtain crude peptides containing several peptides. S3: Using Bruker tims-TOF Pro 2 The peptides were identified using high-resolution ion mobility mass spectrometry (Bruker Daltonics, Germany), and the ISQFYQKWK peptide was selected, as shown in Table 1.

[0037] Table 1. Peptide sources and signal intensities

[0038] Example 3: Solid-phase synthesis of anticoagulant peptide ISQFYQKWK S1: Soak 0.5 mmol of 2-chlorotriphenylmethyl chloride resin in 15 mL of dichloromethane to allow it to swell fully. After removing the dichloromethane from the surface of the 2-chlorotriphenylmethyl chloride resin, clean the resin three times with N,N-dimethylformamide. S2: Using 2-chlorotriphenylmethyl chloride resin as a solid support, 1 mmol Fmoc-Lys (Boc)-OH was dissolved in 5 mL N,N-dimethylformamide, and 2 mmol diisopropylethylamine was added to react fully. The reaction was carried out at room temperature with shaking for 2 h to achieve the initial connection of amino acids with the resin. S3: Add 20 mL of a mixed solution of methanol, diisopropylethylamine and dichloromethane (volume ratio of methanol, diisopropylethylamine and dichloromethane is 2:1:17), and react at room temperature for 0.5 h to block unreacted active sites on the resin; S4: Add a mixed solution of piperidine and N,N-dimethylformamide (volume ratio 1:4), react for 0.5 h to remove the Fmoc protecting group; S5: Following the amino acid sequence ISQFYQKWK (extending from the C-terminus to the N-terminus; S2 has already completed the connection of the first Lys at the C-terminus to the resin; this step sequentially couples the remaining amino acids), take 1 mmol Fmoc-Trp(Boc)-OH, 1 mmol Fmoc-Lys(Boc)-OH, 1 mmol Fmoc-Gln(Trt)-OH, 1 mmol Fmoc-Tyr(tBu)-OH, 1 mmol Fmoc-Phe-OH, 1 mmol Fmoc-Gln(Trt)-OH, 1 mmol Fmoc-Ser(tBu)-OH, and 1 mmol Fmoc-Ile-OH, respectively, and dissolve them in 5 mL N,N-dimethylformamide with 1 mmol benzotriazol-1-yl-oxytripyrrolidinephosphide (PyBOP). Add 2 mmol diisopropylethylamine and react at room temperature for 2-4 minutes. h completes a single coupling; after each coupling, repeat S4, expose the amino group, and proceed to the next round of amino acid coupling until all amino acids are linked according to the target sequence. S6: Add a mixture of trifluoroacetic acid, triisobutylsilane, and deionized water (volume ratio of trifluoroacetic acid, triisobutylsilane, and deionized water is 95:2.5:2.5) to 2-chlorotriphenylmethyl chloride resin, react at room temperature for 2 h, filter the reaction solution to remove resin, collect the filtrate and remove impurities by rotary evaporation, add excess ice-cold ether to precipitate, centrifuge to collect the precipitate, wash twice with ice-cold ether and vacuum dry to obtain crude anticoagulant peptide ISQFYQKWK; S7: The crude anticoagulant peptide ISQFYQKWK was detected by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry to confirm the presence of the target peptide. If the target molecular weight peak was detected, the peptide was purified by semi-preparative high performance liquid chromatography using an aqueous solution containing 0.1% trifluoroacetic acid (mobile phase A) and an acetonitrile solution containing 0.1% trifluoroacetic acid (mobile phase B) as the mobile phase. S8: Collect the target peak components, concentrate them by rotary evaporation, and then perform matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) to confirm the components. Finally, use analytical HPLC to determine the purity.

[0039] Test Example 1: Validation of the structure and purity of the anticoagulant peptide ISQFYQKWK The anticoagulant peptide ISQFYQKWK obtained by solid-phase synthesis and purification in Example 3 was used as the sample to be tested, and its structure and purity were verified by electrospray ionization mass spectrometry (ESI-MS) and reversed-phase high-performance liquid chromatography (RP-HPLC).

[0040] (1) Mass spectrometry structure verification Characterization was performed using electrospray ionization mass spectrometry (ESI-MS), and the detection results are as follows:Figure 1 As shown: In the mass spectrum, the horizontal axis represents the mass-to-charge ratio (m / z); the vertical axis represents the ion signal intensity, with higher peak heights indicating stronger response signals from the corresponding ion. The theoretical molecular weight of the target peptide ISQFYQKWK is approximately 1228 Da. During electrospray ionization, this molecule can bind two protons to form a double-charged ion [M+2H]. 2+ According to the mass-to-charge ratio calculation formula: m / z = (M + 2H) / 2, where M is the molecular weight and H is the mass of the proton (approximately 1 Da), therefore m / z = (1228 + 2) / 2 ≈ 615. This means the peak at position 615 in the mass spectrum corresponds to the double-charged ion peak of the target peptide. The characteristic ion peak in the mass spectrum is consistent with the theoretical double-charged ion mass-to-charge ratio of the target peptide ISQFYQKWK, confirming the successful preparation of the target peptide in this experiment.

[0041] (2) Purity detection by high performance liquid chromatography Purity analysis was performed using reversed-phase HPLC, and the chromatographic results are shown in Table 2. Table 2. HPLC purity test results of anticoagulant peptide ISQFYQKWK

[0042] like Figure 2 As shown, the main peak retention time was 10.532 min, the target peptide peak area accounted for 99.033%, and the impurity content was less than 1%, indicating that the purity of the ISQFYQKWK peptide after solid-phase synthesis and purification was >99%, which meets the requirements of subsequent in vitro anticoagulant activity evaluation experiments.

[0043] Test Example 2: Determination of Anticoagulant Properties of Enzymatic Hydrolysate Activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) are generally important and typical indicators for evaluating the anticoagulant effect of peptides. APTT mainly reflects the activity of the intrinsic coagulation pathway; PT reflects the activity of the extrinsic coagulation pathway; and TT is used to evaluate the process of fibrinogen being converted into fibrin.

[0044] Anticoagulation was determined using the YX2000 fully automated coagulation analyzer from Chuanshiyanxing, along with kits for activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). Experimental plasma was collected from healthy donors. Standard anticoagulants were added during blood collection to prevent coagulation. After mixing, the blood was centrifuged to remove cellular components, and the supernatant plasma was collected as experimental plasma. APTT, PT, and TT reagents were added to the instrument, and 40 μL of the treated plasma was added to each experimental tube. The sample group received 10 μL of enzymatic hydrolysis solution, and the control group received an equal volume of buffer solution. Measurements were performed according to the instrument's operating procedures.

[0045] like Figure 3As shown, compared with the control group, the enzymatic hydrolysate obtained from camel milk casein hydrolysate by neutral protease could prolong the coagulation time of various parameters to varying degrees, indicating that the camel milk casein hydrolysate has an inhibitory effect on the blood coagulation process and exhibits certain anticoagulant activity. Specifically, under the conditions of 2500 U / g enzyme dosage and 30 min hydrolysis time, the camel milk casein hydrolysate showed a better coagulation inhibition effect, with a coagulation inhibition rate of 51.78%; simultaneously, its TT prolongation effect was more significant, indicating that the hydrolysate can interfere with the thrombin-mediated fibrin formation process. Based on the combined results of APTT, PT, TT, and coagulation inhibition rate, neutral protease, 2500 U / g, and 30 min were determined as the optimal enzymatic hydrolysis conditions for preparing anticoagulant peptides from camel milk casein.

[0046] Test Example 3: Determination of Activated Partial Thromboplastin Time, Prothrombin Time, and Thrombin Time The plasma preparation and anticoagulation assay were performed the same as in Test Example 2, except that the test sample was replaced with a solid-phase synthesized camel milk casein anticoagulant peptide sample solution, while the control group still received an equal volume of buffer solution. The camel milk casein anticoagulant peptide ISQFYQKWK was denoted as Peptide A.

[0047] like Figure 4 As shown, after the addition of the peptide sample, compared with the control group, camel milk casein anticoagulant peptide ISQFYQKWK showed varying degrees of prolongation of typical coagulation indicators APTT, PT and TT, indicating that camel milk casein anticoagulant peptide ISQFYQKWK has in vitro anticoagulant activity.

[0048] The APTT results showed that the activated partial thromboplastin time (APTT) was higher after Peptide A treatment than in the control group, indicating that camel milk casein anticoagulant peptide ISQFYQKWK can inhibit the intrinsic coagulation pathway to some extent. The PT results showed that Peptide A had a PT of approximately 14.0 s, while the control group had approximately 12.8 s, indicating that camel milk casein anticoagulant peptide ISQFYQKWK also has a certain intervention effect on the extrinsic coagulation pathway. The TT results showed that camel milk casein anticoagulant peptide ISQFYQKWK had a significant effect on prolonging thrombin time, with Peptide A reaching approximately 22.0 s, indicating that camel milk casein anticoagulant peptide ISQFYQKWK affects thrombin activity and fibrin formation. In summary, camel milk casein anticoagulant peptide ISQFYQKWK has a certain anticoagulant effect.

[0049] Test Example 4: Determination of anticoagulant activity (thrombin inhibition rate) The assay was performed by inhibiting fibrin cross-linking, which is responsible for blood clotting. A 1 g / L fibrinogen solution and a 12 U / mL thrombin solution were prepared using a 0.05 M Tris-HCl buffer solution (pH 7.2) containing 0.12 mM NaCl. 140 μL of fibrinogen solution and 40 μL of peptide sample solution were added to each well of a 96-well plate. After incubation at 37°C for 10 min, the absorbance of the sample blank (SB) was measured. 10 μL of thrombin solution was added to each well to initiate the coagulation reaction. After incubation at 37°C for 10 min, the absorbance of the sample (S) was measured. A control group was prepared by adding 40 μL of Tris-HCl buffer instead of the sample.

[0050] Anticoagulant activity (%) = [(C-CB) - (S-SB)] / (C-CB) × 100%; Where C = absorbance after control incubation, CB = absorbance of control blank, S = absorbance after sample incubation, and SB = absorbance of sample blank. Thrombin inhibitory activity is defined as the antithrombin activity per milligram of peptide, in units of ATU / mg.

[0051] Thrombin inhibitory activity = Ce × Y × Ve / m; Ve-standard thrombin loading volume, unit: μL; Ce-standard thrombin activity, unit: U / ml; Y-inhibition rate, in % m-peptide content, unit: mg.

[0052] The anticoagulant activity of 1 mg of anticoagulant peptide ISQFYQKWK was measured to be 98.87 ATU / mg.

Claims

1. A camel milk casein anticoagulant peptide, characterized in that: The camel milk casein anticoagulant peptide is ISQFYQKWK; The amino acid sequence of the camel milk casein anticoagulant peptide ISQFYQKWK is SEQ ID NO:

1.

2. A method for preparing camel milk casein anticoagulant peptide as described in claim 1, characterized in that, Includes the following steps: S1: Centrifuge raw camel milk to remove the upper fat layer, take the lower clear liquid and adjust the pH to 4.6, centrifuge to collect the precipitate, add -20℃ pre-cooled n-hexane, stir at 4℃ for 24 hours to defatting, and change the liquid several times. After filtering through a Buchner funnel, freeze dry for 24 hours to obtain casein. S2: Dissolve casein in Tris-HCl buffer at pH 8.0 to prepare a casein solution with a concentration of 40 mg / mL. Add neutral protease for enzymatic hydrolysis to obtain the hydrolysate. S3: Dilute the enzymatic hydrolysate to 1-5 mg / mL, acidify with formic acid to pH < 3.0, treat with an Oasis HLB solid-phase extraction desalting column, collect the effluent, concentrate by vacuum centrifugation, and dry to obtain crude polypeptide containing several polypeptides. S4: The peptide was identified using ion mobility high-resolution mass spectrometry, and camel milk casein anticoagulant peptide ISQFYQKWK with high anticoagulant activity was obtained.

3. The method for preparing camel milk casein anticoagulant peptide according to claim 2, characterized in that: In S1, the first centrifugation condition is 7500×g, and the centrifugation time is 20 min; the second centrifugation condition is 9000×g, and the centrifugation time is 20 min; the solid-liquid ratio of the precipitate and n-hexane is 1:10; in S3, the centrifugation condition is 500×g.

4. The method for preparing camel milk casein anticoagulant peptide according to claim 2, characterized in that: In S2, the enzyme dosage is 2500 U / g, the enzymatic hydrolysis temperature is 40℃, and the enzymatic hydrolysis time is 30 min.

5. The method for preparing camel milk casein anticoagulant peptide according to claim 2, characterized in that: In step S3, the OasisHLB solid-phase extraction desalting column treatment and crude peptide collection steps include: S31: Add 1 mL of methanol to the solid phase extraction desalting column and let it flow down by gravity, then discard the effluent; S32: Add 700 µL Buffer B, pressurize and elute the column, repeat twice, and discard the eluent; S33: Add 700 µL Buffer A, pressurize and elute from the column, then discard the eluent; S34: Dilute the enzyme digest sample to twice its volume with Buffer A, load 700 µL of the sample onto the column, allow it to flow down by gravity, and collect the eluent.

6. A solid-phase synthesis method for camel milk casein anticoagulant peptide as described in claim 1, characterized in that, Includes the following steps: S1: Soak 2-chlorotriphenylmethyl chloride resin in dichloromethane to make it fully swollen. After removing the dichloromethane from the surface of the 2-chlorotriphenylmethyl chloride resin, clean the 2-chlorotriphenylmethyl chloride resin with N,N-dimethylformamide. S2: Using the 2-chlorotriphenylmethyl chloride resin treated with S1 as a solid support, Fmoc-amino acids were dissolved in N,N-dimethylformamide, and diisopropylethylamine was added to react fully to achieve the initial connection between amino acids and resin. S3: Add a mixed solution of methanol, diisopropylethylamine and dichloromethane, and react at room temperature for 0.5 h to block unreacted active sites on the resin; S4: Add a mixed solution of piperidine and N,N-dimethylformamide, react for 0.5 h to remove the Fmoc protecting group; S5: Dissolve Fmoc-amino acids and benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate in N,N-dimethylformamide, add diisopropylethylamine, and react at room temperature for 2-4 h to complete the coupling. Repeat S4 in sequence, and then carry out the next round of coupling until all amino acids are linked according to the target sequence. S6: Add a cleaving agent to the coupled 2-chlorotriphenylmethyl chloride resin, react at room temperature for 2 h, filter the reaction solution to remove resin, collect the filtrate and remove impurities by rotary evaporation, add excess ice-cold ether to precipitate, centrifuge to collect the precipitate, wash twice with ice-cold ether and then vacuum dry to obtain crude camel milk casein anticoagulant peptide ISQFYQKWK. S7: The crude camel milk casein anticoagulant peptide was detected by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry to confirm the presence of the target peptide. If the target molecular weight peak was detected, the crude camel milk casein anticoagulant peptide was purified by semi-preparative high performance liquid chromatography using an aqueous solution containing 0.1% trifluoroacetic acid and an acetonitrile solution containing 0.1% trifluoroacetic acid as the mobile phase. S8: Collect the target peak components, concentrate them by rotary evaporation, and then perform matrix-assisted laser desorption / ionization time-of-flight mass spectrometry to confirm the components. Finally, use analytical HPLC to determine the purity.

7. The solid-phase synthesis method of camel milk casein anticoagulant peptide according to claim 6, characterized in that: The amino acids used in preparing the camel milk casein anticoagulant peptide ISQFYQKWK are: Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Trp(Boc)-OH.

8. The solid-phase synthesis method of camel milk casein anticoagulant peptide according to claim 6, characterized in that: In step S3, the volume ratio of methanol, diisopropylethylamine, and dichloromethane is 2:1:17; in step S4, the volume ratio of piperidine and N,N-dimethylformamide is 1:

4.

9. The solid-phase synthesis method of camel milk casein anticoagulant peptide according to claim 6, characterized in that: In step S6, the cutting agent is a mixture of trifluoroacetic acid, triisobutylsilane, and deionized water, wherein the volume ratio of trifluoroacetic acid, triisobutylsilane, and deionized water is 95:2.5:2.

5.

10. The application of the camel milk casein anticoagulant peptide as described in claim 1, characterized in that: The camel milk casein anticoagulant peptide is used to prepare pharmaceutical preparations with antithrombotic function.

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