Extraction method of hirudin, oral preparation containing hirudin and preparation method of oral preparation

Abstract

The invention provides an extraction method of hirudin, an oral preparation containing the hirudin and a preparation method of the oral preparation, and belongs to the technical field of leech processing and traditional Chinese medicine. The hirudin extraction method comprises the following steps: S1, soaking fresh leeches in a freeze-drying protective agent, taking out the leeches, wiping the surfaces of the leeches, pre-freezing the leeches at the temperature of 40 DEG C below zero to 50 DEG C below zero for 1-2 hours, and freeze-drying the leeches at the temperature of 20 DEG C below zero to 30 DEG C below zero for 4-6 hours to obtain freeze-dried leeches; s2, crushing the freeze-dried leeches, adding the crushed leeches into water, adding enzyme and lecithin, performing enzymolysis for 4-6 hours under the conditions that the temperature is 35-40 DEG C and the pH value is 7.5-8.5, and cooling to room temperature to obtain an enzymolysis product; s3, sterilization, ultrafiltration and drying are conducted, and hirudin is obtained. The hirudin extracted by the method is high in activity and high in recovery rate.

Description

Technical Field

[0001] The present application relates to the technical field of leech processing and traditional Chinese medicine, and in particular to a method for extracting hirudin, an oral preparation containing the hirudin, and a preparation method thereof. Background Art

[0002] Leeches, also known as leeches, are a precious traditional Chinese medicinal material. They possess anticoagulant, antithrombotic, lipid-lowering, anti-stasis, swelling-reducing, and detoxifying properties. They are used to treat traumatic injuries, as well as cardiovascular diseases such as cerebral hemorrhage, cirrhosis, hypertension, and hyperlipidemia. The primary pharmacological component of leeches is hirudin, an acidic polypeptide extracted and isolated from leeches and fresh saliva. Hirudin is the most active specific thrombin inhibitor discovered to date, exhibiting potent anticoagulant and antiplatelet aggregation effects. Furthermore, hirudin can prevent tumor cell metastasis. When used in conjunction with chemotherapy and radiotherapy, it can enhance blood flow to tumors and improve therapeutic efficacy, leading to its widespread clinical application.

[0003] Hirudin is a protein. Traditional extraction methods include organic solvent extraction, aqueous extraction, salting-out precipitation, and enzymatic hydrolysis. Aqueous extraction typically involves crude extraction with a sodium chloride solution followed by removal of contaminants with trichloroacetic acid. This method is simple, low-cost, and maintains the activity of the hirudin, but the recovery rate is low. Salting-out precipitation uses ammonium sulfate to reduce the solubility of proteins in the leech stock solution, causing them to precipitate, achieving crude extraction. The hirudin extracted with this method retains high activity, but the recovery rate is also low. Organic solvent extraction typically uses ethanol or acetone as the extraction solvent. Ethanol extraction has a low recovery rate and results in loss of hirudin activity. While acetone extraction offers a higher recovery rate, acetone itself is toxic, and strict temperature control is required during acetone extraction to avoid inactivation of the hirudin due to excessive temperatures. Enzymatic hydrolysis has become a popular extraction method in recent years. Patent CN101332211A discloses a leech extract prepared using a controlled enzymatic hydrolysis method and its preparation method. The leech extract is obtained through enzymatic hydrolysis with trypsin and / or pancreatic enzymes, followed by precipitation with an organic solvent, ultrafiltration, and anion exchange chromatography. This technology only utilizes trypsin and / or pancreatic enzymes, resulting in limited enzymatic hydrolysis efficiency. Patent CN103251926A discloses a leech extract and its preparation method and application. The leech is first enzymatically hydrolyzed with pepsin under acidic conditions, and the enzymatic suspension is then hydrolyzed with trypsin under alkaline conditions. The extract is then refined and purified through ultrafiltration, nanofiltration, and cation exchange gel. The resulting leech extract has a purity exceeding 90%. However, pepsin has a certain effect on hirudin, which can easily lead to inactivation. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the first object of the present application is to provide a method for extracting hirudin, which extracts hirudin through freeze-drying, enzymolysis, and filtration purification steps, and has a high recovery rate and high activity of hirudin.

[0005] The second purpose of the present application is to provide an oral preparation containing hirudin. The surface of the enteric-coated tablet is coated with a coating material, which has excellent sustained-release and anticoagulant effects and can be used to treat and prevent chronic thrombotic diseases, etc., making up for the shortcomings of oral polypeptide drugs.

[0006] The third object of the present application is to provide a method for preparing an oral preparation containing hirudin, which is simple and easy to operate.

[0007] To achieve the above objectives, the technical solutions adopted in this application are as follows: According to one aspect of the present application, a method for extracting hirudin is provided, comprising the following steps: S1. Soak fresh leeches in a freeze-drying protective agent, take them out and wipe the surface dry, then pre-freeze them at -40 to -50°C for 1 to 2 hours, and then freeze-dry them at -20 to -30°C for 4 to 6 hours to obtain freeze-dried leeches; S2, crushing the freeze-dried leeches and adding them to water, adding enzymes and lecithin, performing enzymatic hydrolysis at 35-40° C. and pH 7.5-8.5 for 4-6 hours, cooling to room temperature, and obtaining an enzymatic hydrolysis product; S3. Sterilize, ultrafilter and dry to obtain hirudin.

[0008] Furthermore, in step S1, the lyoprotectant comprises, by weight percentage, 5-10% sucrose, 5-8% trehalose, and the balance is water.

[0009] Furthermore, in step S1, the immersion is carried out at a temperature range of room temperature to 37° C. for 2 to 4 hours. During the immersion process, the weight ratio of the lyoprotectant to the leech is (10 to 20):1.

[0010] The present invention involves soaking leeches in a lyoprotectant before freeze-drying, allowing the lyoprotectant to penetrate the leech's tissues. This facilitates the destruction of cell membranes and other structures during the subsequent freezing process, facilitates the dissolution of hirudin, and improves the recovery rate of hirudin. The lyoprotectant also provides a protective effect during the subsequent freeze-drying process, preventing the conformation of hirudin from being disrupted, thereby ensuring that the resulting hirudin retains a high level of activity. The present invention limits the soaking time of the leeches in the lyoprotectant to avoid insufficient soaking time, which prevents the protectant from penetrating the leech's tissues. Excessive soaking time may cause the dissolution of other components in the leech, which may affect the extraction of hirudin to a certain extent. Experiments have shown that excessively long soaking times reduce the recovery rate of hirudin.

[0011] In the freezing process of this application, pre-freezing at -40°C to -50°C, rather than directly freezing at -20°C to -30°C, helps increase the freezing rate and prevents the formation of large ice crystals within the leech cells due to slow freezing, which would disrupt the natural conformation of hirudin and affect its activity. However, the pre-freezing conditions must be controlled to avoid uneven distribution of ice crystals within the cells, which would affect the extraction of hirudin.

[0012] Furthermore, in step S2, the pulverization is carried out by a low-temperature pulverization method well known in the art, such as low-temperature ultrafine pulverization, low-temperature grinding, etc., to prevent the high temperature during the grinding process from affecting the activity of hirudin; the pulverized particle size is less than 150 mesh, preferably 150-200 mesh, which is conducive to the extraction of hirudin.

[0013] Furthermore, in step S2, the weight ratio of freeze-dried leeches to water is (8-15):1.

[0014] Furthermore, in step S2, the enzyme is selected from alkaline protease and / or trypsin, and the amount of enzyme added is 200-500 U / g, that is, 200-500 U of enzyme is added according to 1 g of substrate freeze-dried leech; when alkaline protease and trypsin are added at the same time, the amount of trypsin is not less than 100 U / g.

[0015] Furthermore, in step S2, the amount of lecithin added is 0.3-0.7% of the weight of the freeze-dried leech.

[0016] This application uses enzymatic hydrolysis to extract hirudin, selecting alkaline protease and trypsin for enzymatic hydrolysis. To ensure enzyme activity and avoid excessively high temperatures that destroy the activity of hirudin, this application chooses to perform enzymatic hydrolysis at 35 to 40°C, which can both meet the enzymatic hydrolysis temperature and maintain a high activity of the resulting hirudin. In addition, it has been found that adding a certain amount of lecithin during the enzymatic hydrolysis process helps to destroy the cell membrane structure of the leech, allowing hirudin to be better dissolved and released into water, and reduces the aggregation of hirudin, thereby reducing the loss of hirudin during subsequent filtration and purification, thereby improving the recovery rate of hirudin. However, experiments have found that excessive addition of lecithin not only does not increase the recovery rate of hirudin, but also easily leads to a decrease in the activity of hirudin. Therefore, this application limits the amount of lecithin used.

[0017] Furthermore, in step S3, the sterilization is carried out by a sterilization method well known in the art, as long as the sterilization temperature is kept below 40° C. to avoid destroying the activity of hirudin. Preferably, ultraviolet irradiation is used for sterilization.

[0018] Furthermore, the ultrafiltration step in step S3 includes: filtering the sterilized enzymatic hydrolysate using a 10 kDa to 30 kDa ultrafiltration membrane and a 1000 Da ultrafiltration membrane in sequence, and collecting the non-permeated liquid.

[0019] The present application first filters the sterilized enzymatic hydrolysate using an ultrafiltration membrane with a molecular weight cutoff of 10kDa to 30kDa to retain the enzyme and large molecular weight impurities such as proteins and peptides. The permeate is then filtered using an ultrafiltration membrane with a molecular weight cutoff of 1000Da to remove impurities such as small molecular weight free amino acids with a molecular weight below 1000Da, thereby obtaining hirudin.

[0020] Furthermore, the drying in step S3 is spray drying or freeze drying. In the present application, the spray drying needs to be controlled at a temperature below 40° C. to avoid destroying the activity of hirudin.

[0021] According to another aspect of the present application, there is provided an oral formulation containing hirudin, comprising a core layer and a coating layer; The core layer comprises hirudin obtained by the above extraction method and pharmaceutically acceptable excipients; The coating layer comprises the following raw materials in parts by weight: 1 to 3 parts of carboxymethyl chitosan, 0.3 to 0.8 parts of sorbitol and 0.1 to 0.2 parts of glyceryl monostearate.

[0022] Furthermore, the weight ratio of the core layer to the coating layer is 100:(4-8).

[0023] Furthermore, the hirudin content in the core layer is ≥50%, for example, 50%, 60%, 70%, 80%, 90%, etc., and may include, but is not limited to, a combination of any one or more of diluents, excipients, fillers, adhesives, wetting agents, and absorption enhancers. It should be noted that the aforementioned diluents, excipients, fillers, adhesives, wetting agents, and absorption enhancers are all commonly used excipients in the art and are therefore not described in detail here. Specifically, in the following specific examples, starch and microcrystalline cellulose are added as excipients.

[0024] Furthermore, the carboxymethyl chitosan has a carboxymethyl substitution degree DS of 0.3 to 0.45 and a molecular weight of 50 kDa to 70 kDa.

[0025] Furthermore, the oral preparation is a tablet or capsule.

[0026] Hirudin, when administered orally, is easily degraded and enzymatically broken down in the gastrointestinal tract, resulting in low bioavailability. However, when administered as an injection, the duration of efficacy is short, and frequent injections cause significant physical and mental distress to patients requiring long-term anticoagulation. Currently, the approach is to coat hirudin with a coating material to resist gastric acid damage, prolong its disintegration time, and allow it to reach the colon for absorption.

[0027] The present application uses carboxymethyl chitosan as the main material of the outer coating layer to wrap hirudin to prepare an oral preparation. By selecting carboxymethyl chitosan with a specific DS and molecular weight, it can be dissolved only in an environment of pH>6.5, that is, in the colon, avoiding premature dissolution in the small intestine, improving colon targeting, and can be quickly degraded by the flora in the colon, disintegrating rapidly, and enabling rapid administration. The resulting oral preparation also has good high-temperature stability, which makes up for the problem that the activity of hirudin is easily destroyed at high temperatures. Although the increase in the DS and molecular weight of carboxymethyl chitosan improves its film-forming properties, an excessively high carboxylmethyl content can easily lead to an increase in its solubility in the intestinal environment, affecting colon targeting, and affecting the effect of the flora in the colon, slowing down the degradation rate in the colon, and also affecting its high-temperature stability; an excessively high molecular weight can also affect its degradation and affect the release of the drug. A reduced molecular weight of carboxymethyl chitosan will lead to poor film-forming properties, easy rupture, and affect targeted drug release and stability. The present application adds a certain amount of sorbitol and glyceryl monostearate to the outer coating layer, which can increase film-forming properties and prevent the outer coating layer from rupturing, thereby improving targeted drug release and high-temperature stability. In addition, the lecithin retained in hirudin also helps spread the coating material, making the film layer more uniform, which helps to improve targeted drug release and high-temperature stability.

[0028] According to another aspect of the present application, a method for preparing an oral preparation containing hirudin is provided, comprising the following steps: Hirudin and pharmaceutically acceptable excipients are mixed to prepare a core layer, 1 to 3 parts of carboxymethyl chitosan, 0.3 to 0.8 parts of sorbitol and 0.1 to 0.2 parts of glyceryl monostearate are added to 100 parts of water to prepare a coating solution, and then the coating layer is applied to the surface of the core layer by a spray coating method, and the weight of the core layer increases by 3 to 7%.

[0029] Furthermore, in the spray coating method, the coating liquid is placed in a fluidized bed coater, the inlet air temperature is controlled to be no higher than 40°C, the outlet air temperature is controlled to be no higher than 35°C, the spray speed is 10-18 rpm, and the spray pressure is adjusted to 0.2-0.3 MPa.

[0030] Furthermore, when the oral preparation is a capsule, it also includes finally filling it into the capsule shell.

[0031] Compared with the prior art, this application has the following beneficial effects: 1. The present application provides a method for extracting hirudin, comprising freeze-drying, enzymatic hydrolysis, and filtration purification steps. By soaking the hirudin in a freeze-drying protectant before freeze-drying and adding lecithin during the enzymatic hydrolysis, the recovery rate of hirudin is high and the activity is high.

[0032] 2. The leech micropowder provided in this application is prepared by coating hirudin with carboxymethyl chitosan. It disintegrates slowly in the stomach and small intestine and quickly in the colon, thereby improving colon targeting and high bioavailability. DETAILED DESCRIPTION

[0033] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present application, but are not intended to limit the present application in any way. The following contents are merely illustrative of the scope of protection claimed in the present application. Those skilled in the art may make various changes and modifications to the invention of the present application based on the disclosed contents, and such changes and modifications shall also fall within the scope of protection claimed in the present application.

[0034] The present application is further described below by way of specific examples. Unless otherwise specified, the various chemical reagents used in the examples of the present application were obtained through conventional commercial channels.

[0035] Example 1 This embodiment provides a method for extracting hirudin, comprising the following steps: S1. Place fresh leeches in 20 times the weight of a freeze-drying protective agent, soak at room temperature for 4 hours, take out and wipe the surface dry, then pre-freeze at -40°C for 2 hours, and then freeze-dry at -20°C for 6 hours to obtain freeze-dried leeches; The lyoprotectant contains: 5% sucrose, 8% trehalose, and the balance is water; S2, grinding the freeze-dried leeches through a 150-mesh sieve and adding them to water, adding 200 U of trypsin and 0.003 g of lecithin per 1 g of freeze-dried leeches, and performing enzymatic hydrolysis at 35°C and pH = 8.5 for 6 h, and cooling to room temperature to obtain a crude extract; S3. Sterilize by ultraviolet irradiation, then filter with a 10kDa ultrafiltration membrane to remove macromolecular substances, then pass the permeate through a 1000Da ultrafiltration membrane, collect the non-permeate liquid as the purified hirudin liquid, and finally spray dry to obtain hirudin.

[0036] Example 2 This embodiment provides another method for extracting hirudin, comprising the following steps: S1. Place fresh leeches in 20 times the weight of a freeze-drying protective agent, soak at 37°C for 2 hours, take out and wipe the surface dry, then pre-freeze at -50°C for 1 hour, and then freeze-dry at -30°C for 4 hours to obtain freeze-dried leeches; The lyoprotectant contains: 10% sucrose, 5% trehalose, and the balance is water; S2, grinding the freeze-dried leeches through a 200-mesh sieve and adding them to water, adding 500 U of alkaline protease and 0.007 g of lecithin to 1 g of freeze-dried leeches, and performing enzymatic hydrolysis at 40°C and pH 7.5 for 6 h, and cooling to room temperature to obtain a crude extract; S3. Sterilize by ultraviolet irradiation, then filter with a 30kDa ultrafiltration membrane to remove macromolecular substances, then pass the permeate through a 1000Da ultrafiltration membrane, collect the non-permeate liquid as the purified hirudin liquid, and finally spray dry to obtain hirudin.

[0037] Example 3 This embodiment provides another method for extracting hirudin, which differs from Example 2 in that, in step S1, the fresh leeches are soaked in the freeze-drying protective agent for 6 hours; the rest is the same as Example 2.

[0038] Example 4 This embodiment provides another method for extracting hirudin, which differs from Example 2 in that in step S1, fresh leeches are not soaked in a lyoprotectant, but are directly mixed with the lyoprotectant and then pre-frozen; the rest is the same as Example 2.

[0039] Example 5 This embodiment provides another method for extracting hirudin, which differs from Example 2 in that the fresh leeches are not soaked in a freeze-drying protective agent in step S1; the rest of the method is the same as Example 2.

[0040] Example 6 This embodiment provides another method for extracting hirudin, which differs from Example 2 in that the pre-freezing temperature in step S1 is -60°C; the rest is the same as Example 2.

[0041] Example 7 This example provides another method for extracting hirudin, which differs from Example 2 in that there is no pre-freezing step in step S1, and the product is directly freeze-dried at -30°C for 4 hours; the rest is the same as Example 2.

[0042] Example 8 This example provides another method for extracting hirudin, which differs from Example 2 in that 0.012 g of lecithin is added during the enzymatic hydrolysis process in step S2; the rest is the same as Example 2.

[0043] Example 9 This example provides another method for extracting hirudin, which differs from Example 2 in that lecithin is not added during the enzymatic hydrolysis process in step S2; the rest is the same as Example 2.

[0044] Example 10 This example provides another method for extracting hirudin, which differs from Example 2 in that there is no pre-freezing step in step S1, and no lecithin is added during the enzymatic hydrolysis process in step S2; the rest is the same as Example 2.

[0045] Test Example 1 This test used a thrombin titration method to test the recovery and activity of hirudin obtained according to the methods provided in Examples 1-10. The principle is based on a 1:1 (mol / mol) ratio of hirudin to thrombin; the International Unit (NIH) for thrombin, and hirudin activity expressed as antithrombin activity units (ATUs); one AUT equals the amount of hirudin required to neutralize one NIH thrombin. Specifically, the hirudin activity A1 in the crude extract obtained in step S2 and the hirudin activity A2 (i.e., the activity of the product hirudin) in the purified hirudin solution obtained in step S3 were measured. The hirudin recovery was calculated using the formula: Recovery = A2 / A1 × 100%.

[0046] The test results are shown in Table 1. It can be seen that the hirudin obtained by the extraction method provided by the present application has high activity and high recovery rate.

[0047] Table 1

[0048] Example 11 This embodiment provides an oral preparation containing leech powder, comprising a core layer and a coating layer; The core layer is the hirudin obtained in Examples 1 to 10 above and pharmaceutically acceptable excipients; The coating layer comprises the following raw materials in parts by weight: 1 to 3 parts of carboxymethyl chitosan, 0.3 to 0.8 parts of sorbitol and 0.1 to 0.2 parts of glyceryl monostearate; The carboxymethyl substitution degree DS of the carboxymethyl chitosan is 0.3-0.45, and the molecular weight is 50 kDa-70 kDa.

[0049] The method for preparing the leech powder comprises the following steps: Hirudin and pharmaceutically acceptable excipients are mixed to prepare a core layer, 1 to 3 parts of carboxymethyl chitosan, 0.3 to 0.8 parts of sorbitol and 0.1 to 0.2 parts of glyceryl monostearate are added to 100 parts of water to prepare a coating solution, and then the coating layer is applied to the surface of the core layer by a spray coating method, and the weight of the core layer increases by 3 to 7%.

[0050] The key point of the oral preparation with colon-targeted drug release is the outer coating. Therefore, in order to verify the colon targeting of the oral preparation of this application, this application uses the weight ratio of hirudin and pharmaceutically acceptable carrier (microcrystalline cellulose and starch fixed at a weight ratio of 2:1) in the core layer as an example to prepare tablets for testing.

[0051] The following tablets were prepared according to the above formulation and method, as follows: Tablets 1#~10# The hirudin in the core layer is the hirudin obtained in Examples 1 to 10 above; The coating layer comprises the following raw materials in parts by weight: 3 parts of carboxymethyl chitosan (DS=0.3, molecular weight 70 kDa), 0.3 parts of sorbitol and 0.1 parts of glyceryl monostearate.

[0052] The hirudin obtained in Example 2 was used, and the coating material formula was the same as that of tablets 1# to 8#.

[0053] Tablets 11#~16# Tablets 11# to 16# use the hirudin obtained in Example 2, and the specific coating layer formula is shown in Table 2 below.

[0054] Table 2

[0055] Test Example 2 The in vitro release properties of the tablets prepared in Example 11 were tested. Specifically, the tablets were first placed in a 0.1 mol / L hydrochloric acid solution (simulating artificial gastric fluid) and sampled after 4 hours. The tablets were then transferred to a pH 6.5 phosphate buffer solution (simulating artificial intestinal fluid) and sampled after 6 hours. Finally, the tablets were transferred to a pH 7.5 phosphate buffer solution (simulating artificial colonic fluid) and sampled after 7 and 8 hours. The cumulative release percentage was calculated, noting that the times above are cumulative.

[0056] The test results are shown in Table 3 below. It can be seen that the oral preparation containing hirudin provided in this application has excellent colon-targeted drug release. Although there is a certain amount of drug release in the gastric juice and intestinal fluid environments of pH 1.2 and pH 6.5, the release amount is very small and can be ignored. Under the condition of pH 7.5, the release amount can reach 90% in 2 hours, that is, it can achieve rapid release in the colon environment, and the drug has good colon targeting. Comparing tablet 2# and tablets 12# to 16#, it can be seen that the DS and molecular weight of carboxymethyl chitosan in the coating need to be limited to a certain range. The DS and molecular weight cooperate to give the coating good colon-targeted drug release. In addition, the addition of sorbitol and glyceryl monostearate to the coating also helps to improve its disintegration performance.

[0057] Table 3

[0058] Test Example 3 Tablets 2#, 8#-10#, and 12#-16# prepared in Example 11 were tested for their stability. The stability of the tablets was tested according to the requirements of the influencing factors test principles in Appendix XIX C of the 2010 edition of the Chinese Pharmacopoeia, Part II, Guidelines for Stability Testing of APIs and Pharmaceutical Preparations. The tablets were placed in culture dishes and placed in thermostats at 40°C and 60°C, respectively. Samples were taken and tested after 5 and 10 days. The results are shown in Table 4 below. It can be seen that the oral preparations prepared by the method described herein exhibit good stability and maintain high antithrombin activity even after high-temperature treatment.

[0059] Table 4

[0060] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, this application is not limited to the above-described embodiments. Any improvements and modifications made by those skilled in the art based on the disclosure of this application, without departing from the scope of this application, should be within the scope of protection of this application.

Claims

1. A method for extracting hirudin, characterized in that: The following steps are involved: S1. Soak fresh leeches in a freeze-drying protective agent, take them out and wipe the surface dry, then pre-freeze them at -40 to -50°C for 1 to 2 hours, and then freeze-dry them at -20 to -30°C for 4 to 6 hours to obtain freeze-dried leeches; S2, crushing the freeze-dried leeches and adding them to water, adding enzymes and lecithin, performing enzymatic hydrolysis at 35-40° C. and pH 7.5-8.5 for 4-6 hours, cooling to room temperature, and obtaining an enzymatic hydrolysis product; S3. Sterilize, ultrafilter and dry to obtain hirudin.

2. The extraction method according to claim 1, wherein In step S1, the lyoprotectant comprises, by weight percentage, 5-10% sucrose, 5-8% trehalose, and the balance is water.

3. The extraction method according to claim 1, wherein In step S1, the soaking is performed in a temperature range from room temperature to 37° C., and the soaking time is 2 to 4 hours.

4. The extraction method according to claim 1, wherein In step S2, the enzyme is selected from alkaline protease and / or trypsin, and the amount of enzyme added is 200 to 500 U / g.

5. The extraction method according to claim 1, wherein In step S2, the amount of lecithin added is 0.3-0.7% of the weight of the freeze-dried leech.

6. The extraction method according to claim 1, characterized in that The ultrafiltration step in step S3 includes filtering the sterilized enzymatic hydrolysate using a 10 kDa to 30 kDa ultrafiltration membrane and a 1000 Da ultrafiltration membrane in sequence, and collecting the non-permeated liquid.

7. An oral preparation containing hirudin, characterized in that: comprising a core layer and a coating layer; The core layer comprises hirudin obtained by the extraction method according to any one of claims 1 to 6 and pharmaceutically acceptable excipients; The coating layer comprises the following raw materials in parts by weight: 1 to 3 parts of carboxymethyl chitosan, 0.3 to 0.8 parts of sorbitol and 0.1 to 0.2 parts of glyceryl monostearate; The weight ratio of the core layer to the coating layer is 100:(4-8).

8. The leech powder according to claim 7, characterized in that The content of hirudin in the core layer is ≥50%.

9. The leech powder according to claim 7, characterized in that The carboxymethyl substitution degree DS of the carboxymethyl chitosan is 0.3-0.45, and the molecular weight is 50-70 kDa.

10. The method for preparing the oral preparation containing hirudin according to any one of claims 7 to 9, characterized in that: The following steps are involved: Hirudin and pharmaceutically acceptable excipients are mixed to prepare a core layer, 1 to 3 parts of carboxymethyl chitosan, 0.3 to 0.8 parts of sorbitol and 0.1 to 0.2 parts of glyceryl monostearate are added to 100 parts of water to prepare a coating solution, and then the coating layer is applied to the surface of the core layer by a spray coating method, and the weight of the core layer increases by 3 to 7%.

Images