Method for efficiently extracting hirudin from poecilobdella manillensis living body

Abstract

The invention discloses a method for efficiently extracting hirudin from poecilobdella manillensis living bodies, and belongs to the technical field of hirudin separation and extraction. The method comprises the following steps: after hunger treatment of poecilobdella manillensis, inducing secretion by combining an induction solution containing malic acid, citric acid, calcium racemic ketoisoleucine and other components with a pig blood extract, and centrifuging to obtain a crude extract; the method comprises the following steps: preparing an immobilized microsphere carrier through L-cysteine functional modification and amino cationic starch crosslinking, immobilizing thrombin, mixing with a crude extract, extracting, eluting, and freeze-drying to obtain a hirudin product. By optimizing an induced secretion system and an immobilized adsorption purification process, the problems of low secretion induction efficiency, complex purification and insufficient activity retention rate in a traditional method are solved, in-vivo repeated extraction of hirudin is realized, and the method has the advantages of high extraction efficiency, high product purity, high specific activity, reutilization of leech living bodies and the like, and is suitable for industrial production. The large-scale production requirement can be met.

Description

Technical Field

[0001] The invention belongs to the technical field of hirudin separation and extraction, and in particular relates to a method for efficiently extracting hirudin from living Hirudo philadelphica. Background Art

[0002] Medicinal leeches have a long history of application in traditional Chinese medicine. They are salty, bitter, and neutral in nature, and have the effects of breaking up blood, removing blood stasis, and promoting menstruation. Modern pharmacological studies have shown that leeches contain a variety of bioactive ingredients, which give them anticoagulant, antithrombotic, lipid-lowering, anti-tumor, anti-apoptotic, and anti-inflammatory effects. They have shown important value in the treatment and prevention of cardiovascular and cerebrovascular diseases (such as arteriosclerosis and cerebral thrombosis), tumors, gout, and kidney disease. Among them, hirudin extracted from leeches is an acidic peptide compound composed of 65-66 amino acids. Its molecular structure contains three pairs of disulfide bonds and is the most active natural thrombin inhibitor known to date. In clinical applications, hirudin has been used to treat disseminated intravascular coagulation (DIC), unstable angina (USA), acute myocardial infarction (AM), and continuous renal replacement therapy (CRRT). Its highly effective anticoagulant and antithrombotic properties provide new ideas for the treatment of related diseases.

[0003] With the increasing demand for natural hirudin in the pharmaceutical industry, how to efficiently extract and purify hirudin has become a research hotspot. At present, the main methods for extracting hirudin include anatomical extraction, induced secretion, and genetic engineering expression. However, the anatomical extraction method requires killing the leech, which not only cannot achieve live reuse, but may also cause hirudin degradation due to the release of proteases due to cell fragmentation; the traditional induced secretion method often uses a single stimulus (such as blood extract) to induce leech secretion, which has problems such as low secretion volume, complex composition, and difficulty in subsequent purification; although genetic engineering methods can achieve recombinant expression of hirudin, there are limitations such as low activity of the expression product and high purification cost. In the existing technology, the method for efficiently extracting hirudin from living leeches still has problems such as low secretion induction efficiency, complex purification process, and insufficient activity retention rate, which makes it difficult to meet the needs of large-scale production. Summary of the Invention

[0004] In view of the above situation, in order to overcome the defects of the existing technology, the present invention provides a method for efficiently extracting hirudin from living leeches. By optimizing the secretion induction system and the immobilized adsorption purification process, the method realizes the repeated extraction of hirudin from living bodies, and solves the problems of low secretion induction efficiency, complex purification process, and insufficient activity retention rate in traditional methods. It has the advantages of high extraction efficiency, high product purity, and reusable living leeches, and can meet the needs of large-scale production.

[0005] In order to achieve the above object, the following technical solution is adopted: The present invention provides a method for efficiently extracting hirudin from living leeches, comprising the following steps:

[0006] S1. Extraction of crude hirudin extract: starve leeches for 20 days, place the leeches in a sealed container, add a prepared induction solution, and incubate at 18-22°C in the dark for 30 minutes to induce the leeches to secrete hirudin. Then, add pig blood extract to the container to stimulate the leeches to continuously secrete hirudin. The secretion fluid is collected for 4 hours, and the collected secretion fluid is centrifuged at 4°C and 12,000 rpm for 20 minutes to remove impurities. The supernatant is collected to obtain a crude hirudin extract.

[0007] S2. Preparation of immobilized microsphere carriers:

[0008] S201, adding L-cysteine ​​and 1-(2-chloro-5-sulfonic acid phenyl)-3-methyl-5-pyrazolone to dimethyl sulfoxide, stirring and reacting at 60° C. for 6 hours. After the reaction, pouring the reaction solution into 3 volumes of ice water to precipitate, filtering, washing the precipitate with ethanol three times, and vacuum drying to obtain sulfonic acid group and pyrazolone functionalized L-cysteine;

[0009] S202, adding functionalized L-cysteine ​​to a reactor, adding ethyl acetate under the protection of nitrogen to obtain a first solution, dissolving triphosgene in ethyl acetate to obtain a second solution, reflux the first solution at 80° C. for 0.5 hour, then adding the second solution dropwise to the first solution, and continuing to reflux at 80° C. for 3 hours. After the reaction is completed, cooling to room temperature, washing with saturated sodium bicarbonate solution and saturated sodium chloride solution alternately twice, extracting and separating the liquid with ethyl acetate, concentrating, and recrystallizing with a mixed solution of ethyl acetate / petroleum ether with a volume twice that of the concentrate to obtain N-carboxyl ring anhydride of functionalized L-cysteine;

[0010] S203, adding the N-carboxyl ring anhydride of functionalized L-cysteine ​​and amino cationic starch to N,N-dimethylformamide, reacting at 40° C. for 24 hours under nitrogen protection, and slowly adding the reaction solution dropwise to 5 volumes of anhydrous ethanol after the reaction, placing the precipitate in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzing the precipitate with deionized water for 48 hours, and freeze-drying the product for 48 hours to obtain functionalized polycysteine-modified amino cationic starch, which is the immobilized microsphere carrier;

[0011] S3. Preparation of immobilized thrombin: adding the immobilized microsphere carrier to a 5% by mass sodium chloride solution, dissolving thrombin in a phosphate buffer solution at a pH of 7.4 to obtain a thrombin solution, adding the thrombin solution to the sodium chloride solution carried by the immobilized microspheres to obtain a final concentration of thrombin in the system of 1 g / L, then adding a 2% by volume glutaraldehyde solution, and reacting the immobilized enzyme at 25° C. for 4 hours. After the reaction is completed, filtering, and washing three times with a phosphate buffer solution at a pH of 7.4 to obtain the immobilized thrombin;

[0012] S4. Mix the crude hirudin extract with immobilized thrombin, and extract by shaking at 25°C and 150 rpm for 2 hours. After the extraction, centrifuge at 4000 rpm for 10 minutes to separate the supernatant, elute the precipitate, collect the eluate, concentrate it to 1 / 10 of the original volume, and lyophilize it at -50°C and 0.01 mbar for 24 hours to obtain the hirudin product.

[0013] Furthermore, the induction solution in step S1 comprises the following components in mass percentage: 0.5-2% malic acid, 0.5-1.5% citric acid, 0.8-1% sodium chloride, 0.5-1.2% glucose, 0.4-0.8% calcium acetophenone isoleucinate, 0.01-0.05% 3-allyloxy-4-methoxybenzaldehyde, 0.05-0.2% sodium oleth-7 phosphate, and the remainder is water.

[0014] Furthermore, the mass ratio of the induction solution to the leech is 3-8:1.

[0015] Furthermore, in step S1, the amount of the pig blood extract added is 5-10% of the mass of the mixed solution, and the pig blood extract is obtained by freeze-drying fresh pig blood and then crushing it into 100 meshes.

[0016] Furthermore, in step S201, the mass ratio of L-cysteine, 1-(2-chloro-5-sulfonic acid phenyl)-3-methyl-5-pyrazolone and dimethyl sulfoxide is 1:1.2-1.8:12-18.

[0017] Furthermore, in step S202, the mass ratio of functionalized L-cysteine ​​to ethyl acetate in the first solution is 1:20-40.

[0018] Furthermore, in step S202, the mass ratio of triphosgene to ethyl acetate in the second solution is 1:8-12.

[0019] Furthermore, in step S202, the mass ratio of L-cysteine ​​to triphosgene is 1:1.1-1.3.

[0020] Furthermore, in step S202, the volume ratio of ethyl acetate to petroleum ether in the ethyl acetate / petroleum ether mixed solution is 1:2-3.

[0021] Furthermore, in step S203, the mass ratio of the functionalized L-cysteine ​​N-carboxyl ring anhydride, amino cationic starch and N,N-dimethylformamide is 3-8:1:40-60.

[0022] In step S3, the mass ratio of the immobilized microsphere carrier to the sodium chloride solution is 1:30-50.

[0023] The concentration of the thrombin solution is 5-10 g / L.

[0024] The amount of glutaraldehyde solution added in step S3 is 1-5% of the total volume of the system.

[0025] Furthermore, in step S4, the crude hirudin extract and the immobilized thrombin are mixed in a volume ratio of 10-20:1.

[0026] Furthermore, the eluent in step S4 is a phosphate buffer solution with a pH of 7.4 and containing 0.5-1.0 mol / L NaCl.

[0027] The beneficial effects of the present invention are:

[0028] (1) A specific combination of induction fluids is used to induce starvation-treated leeches to secrete saliva, wherein racemic ketoisoleucine calcium, as an amino acid derivative, can simulate the nitrogen source in the natural food of leeches, activate the secretion pathway of enzymes related to their digestive system, and regulate cell membrane permeability through calcium signals to promote the release of hirudin from secretory cells. Sodium oleth-7 phosphate, as a non-ionic surfactant, can reduce the surface tension of the secretion fluid, promote the diffusion of hirudin from the secretory gland into the solution, and stabilize the protein structure in the secretion fluid;

[0029] (2) The present invention prepares an immobilized carrier by functionalizing and cross-linking L-cysteine ​​with amino cationic starch. After 1-(2-chloro-5-sulfonic acid phenyl)-3-methyl-5-pyrazolone reacts with L-cysteine, a negatively charged sulfonic acid group and a pyrazolone group with a conjugated structure are introduced on the carrier surface. The sulfonic acid group can adsorb the positively charged hirudin molecules through electrostatic action, thereby improving the adsorption specificity. The conjugated system of pyrazolone can form a π-π stacking effect with the aromatic group of hirudin, thereby enhancing the adsorption affinity. At the same time, its cyclic structure can To provide specific binding sites for hirudin, functionalized L-cysteine ​​forms a polycysteine ​​chain through ring-opening polymerization of N-carboxyl intracyclic anhydride, which is cross-linked with aminocationic starch to form a three-dimensional network structure. The positively charged surface of aminocationic starch can adsorb negatively charged thrombin through electrostatic action. At the same time, the hydrophilicity of its polysaccharide skeleton can maintain the natural conformation of thrombin. The three-dimensional network structure provides a larger specific surface area, making thrombin evenly distributed, increasing the probability of contact with hirudin, and improving adsorption efficiency, thereby obtaining a hirudin product with higher yield and higher purity.

[0030] (3) The immobilized thrombin of the present invention has a high adsorption capacity for hirudin. After adsorption, the ionic strength of sodium chloride destroys the electrostatic binding between thrombin and hirudin, thereby achieving a high extraction rate of hirudin. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the appendix in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0033] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and the experimental materials used in the following examples, unless otherwise specified, are all purchased from commercial channels.

[0034] Example 1: A method for efficiently extracting hirudin from living leeches, comprising the following steps:

[0035] S1. Extraction of crude hirudin extract: starve leeches for 20 days, place the leeches in a sealed container, add the prepared induction solution, and stand in the dark at 18°C ​​for 30 minutes to induce the leeches to secrete hirudin, wherein the induction solution comprises the following components in percentage by weight: malic acid 0.5%, citric acid 0.5%, sodium chloride 0.8%, glucose 0.5%, racemic ketoisoleucine calcium 0.4%, 3-allyloxy-4-methoxybenzaldehyde 0.01%, oleth-7 phosphate The invention further comprises the steps of: adding 0.05% sodium ester of hydroxybenzoate and the rest being water, wherein the mass ratio of the induction solution to the leech is 3:1; then adding pig blood extract to the container to stimulate the leech to continue secreting, wherein the amount of the pig blood extract added is 5% of the mass of the mixed solution, and the pig blood extract is obtained by freeze-drying fresh pig blood and then crushing it to 100 mesh; collecting the secretion liquid for 4 hours, centrifuging the collected secretion liquid at 4°C and 12000 rpm for 20 minutes to remove impurities, and collecting the supernatant to obtain a crude hirudin extract;

[0036] S2. Preparation of immobilized microsphere carriers:

[0037] S201, adding L-cysteine ​​and 1-(2-chloro-5-sulfonic acid phenyl)-3-methyl-5-pyrazolone to dimethyl sulfoxide, stirring and reacting at 60° C. for 6 hours, wherein the mass ratio of L-cysteine, 1-(2-chloro-5-sulfonic acid phenyl)-3-methyl-5-pyrazolone and dimethyl sulfoxide is 1:1.2:12; after the reaction, pouring the reaction solution into 3 volumes of ice water to precipitate, filtering, washing the precipitate with ethanol 3 times, and vacuum drying to obtain sulfonic acid group and pyrazolone functionalized L-cysteine;

[0038] S202, adding functionalized L-cysteine ​​to a reactor, adding ethyl acetate under the protection of nitrogen to obtain a first solution, wherein the mass ratio of functionalized L-cysteine ​​to ethyl acetate in the first solution is 1:20; dissolving triphosgene in ethyl acetate to obtain a second solution, wherein the mass ratio of triphosgene to ethyl acetate in the second solution is 1:8; the mass ratio of L-cysteine ​​to triphosgene is 1:1.1; reflux the first solution at 80°C for 0.5 hour, then add the second solution dropwise to the first solution, and continue to reflux at 80°C for 3 hours. After the reaction is completed, cool to room temperature, wash with saturated sodium bicarbonate solution and saturated sodium chloride solution alternately twice, extract and separate the liquid with ethyl acetate, concentrate, and recrystallize with ethyl acetate / petroleum ether mixed solution with a volume twice that of the concentrate, wherein the volume ratio of ethyl acetate to petroleum ether in the ethyl acetate / petroleum ether mixed solution is 1:2; obtain N-carboxyl ring anhydride of functionalized L-cysteine;

[0039] S203, adding the N-carboxyl ring anhydride of functionalized L-cysteine ​​and aminocationic starch to N,N-dimethylformamide, and reacting at 40° C. for 24 hours under nitrogen protection, wherein the mass ratio of the N-carboxyl ring anhydride of functionalized L-cysteine, aminocationic starch, and N,N-dimethylformamide is 3:1:40; after the reaction, slowly adding the reaction solution dropwise to 5 volumes of anhydrous ethanol, precipitating the precipitate and placing it in a dialysis bag with a molecular weight cutoff of 3500Da, dialyzing it with deionized water for 48 hours, and freeze-drying the product for 48 hours to obtain functionalized polycysteine-modified aminocationic starch, which is the immobilized microsphere carrier;

[0040] S3. Preparation of immobilized thrombin: adding the immobilized microsphere carrier to a 5% sodium chloride solution by mass, wherein the mass ratio of the immobilized microsphere carrier to the sodium chloride solution is 1:30; dissolving thrombin in a phosphate buffer solution with a pH value of 7.4 to obtain a thrombin solution, wherein the concentration of the thrombin solution is 5 g / L; adding the thrombin solution to the sodium chloride solution carried by the immobilized microspheres to obtain a final concentration of thrombin in the system of 1 g / L, and then adding a 1% by volume glutaraldehyde solution, wherein the amount of the glutaraldehyde solution added is 1% of the total volume of the system; carrying out the immobilized enzyme reaction at 25° C. for 4 hours. After the reaction is completed, filtering, and washing three times with a phosphate buffer solution with a pH value of 7.4 to obtain the immobilized thrombin;

[0041] S4. Mix the crude hirudin extract with immobilized thrombin in a volume ratio of 10:1; perform oscillation extraction at 25°C and 150 rpm for 2 hours. After the extraction, centrifuge at 4000 rpm for 10 minutes to separate the supernatant, and elute the precipitate with the eluent being a phosphate buffer solution with a pH of 7.4 containing 0.5 mol / L NaCl; collect the eluate, concentrate it to 1 / 10 of its original volume, and lyophilize it at -50°C and 0.01 mbar for 24 hours to obtain the hirudin product.

[0042] Example 2: A method for efficiently extracting hirudin from living leeches, comprising the following steps:

[0043] S1. Extraction of crude hirudin extract: starve leeches for 20 days, place the leeches in a sealed container, add the prepared induction solution, and stand in the dark at 22°C for 30 minutes to induce the leeches to secrete hirudin, wherein the induction solution comprises the following components in mass percentage: 2% malic acid, 1.5% citric acid, 1% sodium chloride, 1.2% glucose, 0.8% calcium hydroxybenzoate, 0.05% 3-allyloxy-4-methoxybenzaldehyde, and oleth-7 phosphate. 0.2% sodium, the remainder being water, the mass ratio of the induction solution to the leech being 8:1; then adding pig blood extract to the container to stimulate the leech to continue secreting, the amount of the pig blood extract added being 10% of the mass of the mixed solution, the pig blood extract being obtained by freeze-drying fresh pig blood and then crushing it to 100 mesh; collecting the secretion fluid for 4 hours, centrifuging the collected secretion fluid at 4°C and 12000 rpm for 20 minutes to remove impurities, and collecting the supernatant to obtain a crude hirudin extract;

[0044] S2. Preparation of immobilized microsphere carriers:

[0045] S201, adding L-cysteine ​​and 1-(2-chloro-5-sulfonic acid phenyl)-3-methyl-5-pyrazolone to dimethyl sulfoxide, stirring and reacting at 60° C. for 6 hours, wherein the mass ratio of L-cysteine, 1-(2-chloro-5-sulfonic acid phenyl)-3-methyl-5-pyrazolone and dimethyl sulfoxide is 1:1.8:18; after the reaction, pouring the reaction solution into 3 volumes of ice water to precipitate, filtering, washing the precipitate with ethanol 3 times, and vacuum drying to obtain sulfonic acid group and pyrazolone functionalized L-cysteine;

[0046] S202, adding functionalized L-cysteine ​​to a reactor, adding ethyl acetate under the protection of nitrogen to obtain a first solution, wherein the mass ratio of functionalized L-cysteine ​​to ethyl acetate in the first solution is 1:40; dissolving triphosgene in ethyl acetate to obtain a second solution, wherein the mass ratio of triphosgene to ethyl acetate in the second solution is 1:12; the mass ratio of L-cysteine ​​to triphosgene is 1:1.3; reflux the first solution at 80°C for 0.5 hour, then add the second solution dropwise to the first solution, and continue to reflux at 80°C for 3 hours. After the reaction is completed, cool to room temperature, wash with saturated sodium bicarbonate solution and saturated sodium chloride solution alternately twice, extract and separate the liquid with ethyl acetate, concentrate, and recrystallize with ethyl acetate / petroleum ether mixed solution with a volume twice that of the concentrate, wherein the volume ratio of ethyl acetate to petroleum ether in the ethyl acetate / petroleum ether mixed solution is 1:3; obtain N-carboxyl ring anhydride of functionalized L-cysteine;

[0047] S203, adding the N-carboxyl ring anhydride of functionalized L-cysteine ​​and aminocationic starch to N,N-dimethylformamide, and reacting at 40° C. for 24 hours under nitrogen protection, wherein the mass ratio of the N-carboxyl ring anhydride of functionalized L-cysteine, aminocationic starch, and N,N-dimethylformamide is 8:1:60; after the reaction, slowly adding the reaction solution dropwise to 5 volumes of anhydrous ethanol, and placing the precipitate into a dialysis bag with a molecular weight cutoff of 3500Da, dialyzing with deionized water for 48 hours, and freeze-drying the product for 48 hours to obtain functionalized polycysteine-modified aminocationic starch, which is the immobilized microsphere carrier;

[0048] S3. Preparation of immobilized thrombin: adding the immobilized microsphere carrier to a 5% sodium chloride solution by mass, wherein the mass ratio of the immobilized microsphere carrier to the sodium chloride solution is 1:50; dissolving thrombin in a phosphate buffer solution with a pH value of 7.4 to obtain a thrombin solution, wherein the concentration of the thrombin solution is 10 g / L; adding the thrombin solution to the sodium chloride solution carried by the immobilized microspheres to obtain a final concentration of thrombin in the system of 1 g / L, and then adding a 5% by volume glutaraldehyde solution, wherein the amount of the glutaraldehyde solution added is 5% of the total volume of the system; carrying out the immobilized enzyme reaction at 25° C. for 4 hours. After the reaction is completed, filtering, and washing three times with a phosphate buffer solution with a pH value of 7.4 to obtain the immobilized thrombin;

[0049] S4. Mix the crude hirudin extract with immobilized thrombin in a volume ratio of 20:1; perform oscillation extraction at 25°C and 150 rpm for 2 hours. After the extraction, centrifuge at 4000 rpm for 10 minutes to separate the supernatant, and elute the precipitate with the eluent being a phosphate buffer solution with a pH of 7.4 containing 1.0 mol / L NaCl; collect the eluate, concentrate it to 1 / 10 of its original volume, and lyophilize it at -50°C and 0.01 mbar for 24 hours to obtain the hirudin product.

[0050] Example 3: A method for efficiently extracting hirudin from living leeches, comprising the following steps:

[0051] S1. Extraction of crude hirudin extract: starve leeches for 20 days, place the leeches in a sealed container, add a prepared induction solution, and stand in the dark at 20°C for 30 minutes to induce the leeches to secrete hirudin, wherein the induction solution comprises the following components in percentage by mass: 1.3% malic acid, 1.0% citric acid, 0.9% sodium chloride, 0.9% glucose, 0.6% calcium hydroxybenzoate, 0.03% 3-allyloxy-4-methoxybenzaldehyde, 0.03% oleth-7 phosphate 0.12% sodium ester, the remainder being water, the mass ratio of the induction solution to the leech being 5:1; then adding pig blood extract to the container to stimulate the leech to continue secreting, the amount of the pig blood extract added being 7.5% of the mass of the mixed solution, the pig blood extract being obtained by freeze-drying fresh pig blood and then crushing it to 100 mesh; collecting the secretion fluid for 4 hours, centrifuging the collected secretion fluid at 4°C and 12000 rpm for 20 minutes to remove impurities, and collecting the supernatant to obtain a crude hirudin extract;

[0052] S2. Preparation of immobilized microsphere carriers:

[0053] S201, adding L-cysteine ​​and 1-(2-chloro-5-sulfonic acid phenyl)-3-methyl-5-pyrazolone to dimethyl sulfoxide, stirring and reacting at 60° C. for 6 hours, wherein the mass ratio of L-cysteine, 1-(2-chloro-5-sulfonic acid phenyl)-3-methyl-5-pyrazolone and dimethyl sulfoxide is 1:1.5:15; after the reaction, pouring the reaction solution into 3 volumes of ice water to precipitate, filtering, washing the precipitate with ethanol 3 times, and vacuum drying to obtain sulfonic acid group and pyrazolone functionalized L-cysteine;

[0054] S202, adding functionalized L-cysteine ​​to a reactor, adding ethyl acetate under the protection of nitrogen to obtain a first solution, wherein the mass ratio of functionalized L-cysteine ​​to ethyl acetate in the first solution is 1:30; dissolving triphosgene in ethyl acetate to obtain a second solution, wherein the mass ratio of triphosgene to ethyl acetate in the second solution is 1:10; the mass ratio of L-cysteine ​​to triphosgene is 1:1.2; the first solution is refluxed at 80°C for 0.5 hour, and then the second solution is added dropwise to the first solution, and the reflux reaction is continued at 80°C for 3 hours. After the reaction is completed, it is cooled to room temperature, and the solution is washed alternately with saturated sodium bicarbonate solution and saturated sodium chloride solution twice, extracted with ethyl acetate, concentrated, and recrystallized with ethyl acetate / petroleum ether mixed solution with a volume twice that of the concentrate, wherein the volume ratio of ethyl acetate to petroleum ether in the ethyl acetate / petroleum ether mixed solution is 1:2.5; to obtain the N-carboxyl ring anhydride of functionalized L-cysteine;

[0055] S203, adding the N-carboxyl ring anhydride of functionalized L-cysteine ​​and aminocationic starch to N,N-dimethylformamide, and reacting at 40° C. for 24 hours under nitrogen protection, wherein the mass ratio of the N-carboxyl ring anhydride of functionalized L-cysteine, aminocationic starch, and N,N-dimethylformamide is 5.5:1:50; after the reaction, slowly adding the reaction solution dropwise to 5 volumes of anhydrous ethanol, and placing the precipitate into a dialysis bag with a molecular weight cutoff of 3500Da, dialyzing with deionized water for 48 hours, and freeze-drying the product for 48 hours to obtain functionalized polycysteine-modified aminocationic starch, which is the immobilized microsphere carrier;

[0056] S3. Preparation of immobilized thrombin: adding the immobilized microsphere carrier to a 5% sodium chloride solution by mass, wherein the mass ratio of the immobilized microsphere carrier to the sodium chloride solution is 1:40; dissolving thrombin in a phosphate buffer solution with a pH value of 7.4 to obtain a thrombin solution, wherein the concentration of the thrombin solution is 7.5 g / L; adding the thrombin solution to the sodium chloride solution carried by the immobilized microspheres to obtain a final concentration of thrombin in the system of 1 g / L, and then adding a glutaraldehyde solution with a volume fraction of 3%, wherein the amount of the glutaraldehyde solution added is 3% of the total volume of the system; carrying out the immobilized enzyme reaction at 25° C. for 4 hours. After the reaction is completed, filtering, and washing three times with a phosphate buffer solution with a pH value of 7.4 to obtain the immobilized thrombin;

[0057] S4. Mix the crude hirudin extract with immobilized thrombin in a volume ratio of 15:1; perform oscillation extraction at 25°C and 150 rpm for 2 hours. After the extraction, centrifuge at 4000 rpm for 10 minutes to separate the supernatant, and elute the precipitate with the eluent being a phosphate buffer solution with a pH of 7.4 containing 0.75 mol / L NaCl; collect the eluate, concentrate it to 1 / 10 of its original volume, and lyophilize it at -50°C and 0.01 mbar for 24 hours to obtain the hirudin product.

[0058] Example 4: A method for efficiently extracting hirudin from live leeches. This example differs from Example 3 only in that the induction solution comprises the following components in percentage by weight: 0.5% malic acid, 0.5% citric acid, 0.9% sodium chloride, 0.5% glucose, 0.4% calcium racemic ketoisoleucinate, 0.05% 3-allyloxy-4-methoxybenzaldehyde, 0.05% sodium oleth-7 phosphate, and the remainder is water. The remaining components and preparation process are the same as those in Example 3.

[0059] Example 5: A method for efficiently extracting hirudin from live leeches. This example differs from Example 3 only in that the induction solution comprises the following components in percentage by weight: 0.5% malic acid, 0.5% citric acid, 0.9% sodium chloride, 0.5% glucose, 0.4% calcium racemic ketoisoleucinate, 0.01% 3-allyloxy-4-methoxybenzaldehyde, 0.2% sodium oleth-7 phosphate, and the remainder is water. The remaining components and preparation process are the same as those in Example 3.

[0060] Comparative Example 1: The only difference between this comparative example and Example 3 is that the induction solution includes the following components in mass percentage: 1.3% malic acid, 1.0% citric acid, 0.9% sodium chloride, 0.9% glucose, and the rest is water; the remaining components and preparation process are the same as those in Example 3.

[0061] Comparative Example 2: The only difference between this comparative example and Example 3 is that unmodified amino cationic starch is directly used as a carrier. The specific operation is: the amino cationic starch is dispersed in a sodium chloride solution with a mass fraction of 5%, and thrombin is immobilized according to the same parameters as in Example 3; the remaining components and preparation process are the same as in Example 3.

[0062] Comparative Example 3: In this comparative example, crude hirudin extract was extracted and separated by integrated chromatography. The specific operation was as follows: the crude hirudin extract was loaded onto a Heparin-Sepharose affinity chromatography column (5 mL column volume), gradient eluted with a pH 7.4 phosphate buffer solution containing 0.5 mol / L NaCl, and the active peak was collected and freeze-dried.

[0063] Comparative Example 4: The crude hirudin extract was mixed with free thrombin in the same proportion as in Example 3, extracted at 25° C. for 2 hours under shaking, centrifuged, and the supernatant was lyophilized; the remaining components and preparation process were the same as in Example 3.

[0064] Test Example: The key indicators of hirudin obtained from each group of Examples and Comparative Examples were tested, including hirudin yield, specific activity, anticoagulant activity and purity. The specific method is as follows:

[0065] The hirudin content in the final hirudin product was determined by high-performance liquid chromatography (HPLC), and the yield was calculated. The procedure was as follows: an Agilent 1100 HPLC system equipped with a preparative C18 column (3.5 mm × 20 cm) was used. The final hirudin product to be measured was dissolved in an appropriate amount of solvent and loaded directly onto the column. The column was first equilibrated with 0.1% trifluoroacetic acid (TFA) in water. Elution was performed using a linear gradient program: the acetonitrile ratio was linearly increased from 0% to 60% over 30 minutes (mobile phase A: 0.1% TFA in water; mobile phase B: 0.1% TFA in acetonitrile) at a flow rate of 1.2 mL / min. The elution process was monitored by ultraviolet light at a wavelength of 215 nm, and fractions corresponding to the hirudin activity peak were collected. The yield was calculated by comparing the total amount of hirudin measured in the final product with the total amount of hirudin in the initial crude hirudin extract.

[0066] Yield (%) = (total amount of hirudin in the final product / total amount of hirudin in the crude extract) × 100%.

[0067] Purity (%) = (area of ​​main hirudin peak / total area of ​​all chromatographic peaks) × 100%.

[0068] The total protein content was determined by the biuret method: 6 test tubes were taken and numbered 0-5, and the components were added according to the following proportions: 0.5 mL of distilled water and 4.0 mL of standard biuret reagent were added to test tube No. 0, 0.1 mL of BSA standard solution, 0.4 mL of distilled water and 4.0 mL of standard biuret reagent were added to test tube No. 1, 0.2 mL of BSA standard solution, 0.3 mL of distilled water and 4.0 mL of standard biuret reagent were added to test tube No. 2, 0.4 mL of BSA standard solution, 0.2 mL of distilled water and 4.0 mL of standard biuret reagent were added to test tube No. 3, 0.2 mL of BSA standard solution, 0.5 mL of distilled water and 4.0 mL of standard biuret reagent were added to test tube No. 4, and 0.5 mL of BSA standard solution and 4.0 mL of standard biuret reagent were added to test tube No. 5. After shaking, the mixture was kept in a 37°C water bath for 30 minutes and then cooled to room temperature. Zero the tube with a blank tube and measure the absorbance of each tube at 540 nm. Plot a standard curve with protein concentration (mg / mL) as the abscissa and absorbance as the ordinate to obtain a linear equation. Next, weigh 0.1 g of hirudin sample, dissolve it in PBS, and dilute to 10 mL. Add 0.5 mL of the diluted sample solution to 4.0 mL of biuret reagent, shake well, and incubate in a 37°C water bath for 30 minutes. After cooling, measure the absorbance at 540 nm. Substitute the sample into the standard curve equation to calculate the protein concentration (C, mg / mL) in the sample solution.

[0069] Total protein content (mg / g sample) = C × dilution factor × constant volume (mL) ÷ sample mass (g).

[0070] The hirudin antithrombin activity units were determined by the chromogenic substrate method, and the specific activity was calculated by the following formula:

[0071] Specific activity = antithrombin activity unit (IU) ÷ total protein content (mg).

[0072] As can be seen from Table 1, in terms of yield, the present invention promotes secretion of leeches by using a specific induction solution, such as calcium racemic ketoisoleucine to simulate a nitrogen source to activate the secretion pathway, and sodium oleth-7 phosphate to promote the diffusion of hirudin. Combined with the pig blood extract to stimulate continuous secretion, the hirudin yield is significantly improved compared to traditional methods, with the yield of each embodiment reaching 29.5-31.8 mg / g leech. In terms of purity, the present invention uses immobilized microsphere carriers to specifically bind to hirudin through sulfonic acid group electrostatic adsorption and pyrazolone π-π stacking, effectively eliminating impurities. The purity of each embodiment is as high as 95.2%-97.8%. In addition, the protein content is also higher than that of the comparative example, and the specific activity is significantly enhanced. The three-dimensional network structure of the immobilized microsphere carrier maintains the natural conformation of thrombin and enhances the binding specificity with hirudin. Overall, the present invention achieves comprehensive improvements in the extraction yield, purity, and activity of hirudin.

[0073] Table 1: Key index test results of hirudin

[0074] project Yield (mg / g leech) Protein content (mg / g sample) Specific activity (IU / mg) purity(%) Example 1 29.5 92.3 15200 95.2 Example 2 30.1 88.7 15800 96.5 Example 3 31.8 85.4 16500 97.8 Example 4 29.7 90.1 15500 95.8 Example 5 31.2 89.9 16100 96.9 Comparative Example 1 18.3 75.6 8300 78.5 Comparative Example 2 22.6 80.7 10200 85.2 Comparative Example 3 26.4 65.8 13800 91.7 Comparative Example 4 24.1 78.2 7600 72.3

[0075] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

[0076] The above description of the present invention and its embodiments is non-limiting and its practical applications are not limited thereto. In short, if a person skilled in the art is inspired by the above description and designs similar methods and embodiments to the technical solution without departing from the purpose of the present invention, they shall fall within the scope of protection of the present invention.

Claims

1. A method for efficiently extracting hirudin from living leeches, characterized in that: The following steps are involved: S1. Extraction of crude hirudin extract: starve leeches for 20 days, place the leeches in a sealed container, add a prepared induction solution, and incubate at 18-22°C in the dark for 30 minutes to induce the leeches to secrete hirudin. Then, add pig blood extract to the container to stimulate the leeches to continuously secrete hirudin. The secretion fluid is collected for 4 hours, and the collected secretion fluid is centrifuged at 4°C and 12,000 rpm for 20 minutes to remove impurities. The supernatant is collected to obtain a crude hirudin extract. S2. Preparation of immobilized microsphere carriers: S201, adding L-cysteine ​​and 1-(2-chloro-5-sulfonic acid phenyl)-3-methyl-5-pyrazolone to dimethyl sulfoxide, stirring and reacting at 60° C. for 6 hours. After the reaction, pouring the reaction solution into 3 volumes of ice water to precipitate, filtering, washing the precipitate with ethanol three times, and vacuum drying to obtain sulfonic acid group and pyrazolone functionalized L-cysteine; S202, adding functionalized L-cysteine ​​to a reactor, adding ethyl acetate under the protection of nitrogen to obtain a first solution, dissolving triphosgene in ethyl acetate to obtain a second solution, reflux the first solution at 80° C. for 0.5 hour, then adding the second solution dropwise to the first solution, and continuing to reflux at 80° C. for 3 hours. After the reaction is completed, cooling to room temperature, washing with saturated sodium bicarbonate solution and saturated sodium chloride solution alternately twice, extracting and separating the liquid with ethyl acetate, concentrating, and recrystallizing with a mixed solution of ethyl acetate / petroleum ether with a volume twice that of the concentrate to obtain N-carboxyl ring anhydride of functionalized L-cysteine; S203, adding the N-carboxyl ring anhydride of functionalized L-cysteine ​​and amino cationic starch to N,N-dimethylformamide, reacting at 40° C. for 24 hours under nitrogen protection, and slowly adding the reaction solution dropwise to 5 volumes of anhydrous ethanol after the reaction, placing the precipitate in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzing the precipitate with deionized water for 48 hours, and freeze-drying the product for 48 hours to obtain functionalized polycysteine-modified amino cationic starch, which is the immobilized microsphere carrier; S3. Preparation of immobilized thrombin: adding the immobilized microsphere carrier to a 5% by mass sodium chloride solution, dissolving thrombin in a phosphate buffer solution at a pH of 7.4 to obtain a thrombin solution, adding the thrombin solution to the sodium chloride solution carried by the immobilized microspheres to obtain a final concentration of thrombin in the system of 1 g / L, then adding a 2% by volume glutaraldehyde solution, and reacting the immobilized enzyme at 25° C. for 4 hours. After the reaction is completed, filtering, and washing three times with a phosphate buffer solution at a pH of 7.4 to obtain the immobilized thrombin; S4. Mix the crude hirudin extract with immobilized thrombin, and extract by shaking at 25°C and 150 rpm for 2 hours. After the extraction, centrifuge at 4000 rpm for 10 minutes to separate the supernatant, elute the precipitate, collect the eluate, concentrate it to 1 / 10 of the original volume, and lyophilize it at -50°C and 0.01 mbar for 24 hours to obtain the hirudin product.

2. The method for efficiently extracting hirudin from living leeches of claim 1, wherein: The induction solution in step S1 includes the following components in mass percentage: 0.5-2% malic acid, 0.5-1.5% citric acid, 0.8-1% sodium chloride, 0.5-1.2% glucose, 0.4-0.8% racemic ketoisoleucine calcium, 0.01-0.05% 3-allyloxy-4-methoxybenzaldehyde, 0.05-0.2% sodium oleth-7 phosphate, and the rest is water. The mass ratio of the induction solution to the leech is 3-8:

1.

3. The method for efficiently extracting hirudin from living leeches of claim 2, wherein: The amount of the pig blood extract added in step S1 is 5-10% of the mass of the mixed solution. The pig blood extract is obtained by freeze-drying fresh pig blood and then crushing it into 100 meshes.

4. The method for efficiently extracting hirudin from living leeches of claim 3, wherein: In step S201, the mass ratio of L-cysteine, 1-(2-chloro-5-sulfonic acid phenyl)-3-methyl-5-pyrazolone and dimethyl sulfoxide is 1:1.2-1.8:12-18.

5. The method for efficiently extracting hirudin from living leeches of claim 4, wherein: In step S202, the mass ratio of functionalized L-cysteine ​​to ethyl acetate in the first solution is 1:20-40; In step S202, the mass ratio of triphosgene to ethyl acetate in the second solution is 1:8-12; In step S202, the mass ratio of L-cysteine ​​to triphosgene is 1:1.1-1.

3.

6. The method for efficiently extracting hirudin from living leeches of claim 5, characterized in that: In step S202, the volume ratio of ethyl acetate to petroleum ether in the ethyl acetate / petroleum ether mixed solution is 1:2-3.

7. The method for efficiently extracting hirudin from living leeches of claim 6, wherein: In the step S203, the mass ratio of the functionalized L-cysteine ​​N-carboxyl ring anhydride, amino cationic starch and N,N-dimethylformamide is 3-8:1:40-60.

8. The method for efficiently extracting hirudin from living leeches of claim 7, wherein: In step S3, the mass ratio of the immobilized microsphere carrier to the sodium chloride solution is 1:30-50, the concentration of the thrombin solution is 5-10 g / L, and the amount of the glutaraldehyde solution added in step S3 is 1-5% of the total volume of the system.

9. The method for efficiently extracting hirudin from living leeches of claim 8, characterized in that: In step S4, the crude hirudin extract and the immobilized thrombin are mixed in a volume ratio of 10-20:1; The eluent in step S4 is a phosphate buffer solution with a pH of 7.4 and containing 0.5-1.0 mol / L NaCl.