An extraction method of hirudin, an oral preparation containing the hirudin and a preparation method thereof

Abstract

The application 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 fields of hirudo processing and traditional Chinese medicine. The extraction method of the hirudin comprises the following steps: S1, soaking fresh hirudo in a freeze-drying protective agent, wiping the surface after taking out, pre-freezing at-40 to-50 DEG C for 1 to 2 hours, and then freeze-drying at-20 to-30 DEG C for 4 to 6 hours to obtain freeze-dried hirudo; S2, crushing the freeze-dried hirudo, adding water, adding enzymes and lecithin, and enzymolysis at 35 to 40 DEG C and pH=7.5 to 8.5 for 4 to 6 hours, and then cooling to room temperature to obtain an enzymolysis product; and S3, sterilizing, ultrafiltering and drying to obtain the hirudin. The hirudin obtained by the method has high activity and high recovery rate.

Description

TECHNICAL FIELD

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

[0002] Leech, also known as "Miaohuang", is a traditional Chinese medicinal material with the functions of anticoagulation, antithrombosis, reducing blood fat, removing food retention and blood stasis, and detoxification, which can be used for treating contusions and injuries, cerebral hemorrhage, liver cirrhosis, hypertension, hyperlipidemia and other cardiovascular diseases. The chemical composition of leech that plays a pharmacological role is mainly hirudin, which is an acidic polypeptide extracted from leeches and fresh saliva, and is the most active specific thrombin inhibitor discovered so far, with good anticoagulant and anti-platelet aggregation effects. In addition, hirudin can also prevent tumor cell metastasis, and can enhance the efficacy of chemotherapy and radiotherapy by promoting blood flow in tumors, and thus is widely used in clinical practice.

[0003] Hirudin is a protein, and the traditional extraction methods mainly include organic solvent extraction, water extraction, salt precipitation and enzymatic hydrolysis. The water extraction method generally uses sodium chloride aqueous solution for crude extraction, and then removes impure proteins with trichloroacetic acid. This method is simple, low in cost, and can ensure the activity of hirudin, but the recovery rate is low. The salt precipitation method uses ammonium sulfate to precipitate the proteins in the leech stock solution according to their solubility, so as to achieve the purpose of crude extraction. The hirudin extracted by this method retains high activity, but the recovery rate is also low. The organic solvent extraction method generally uses ethanol or acetone as the extraction solvent. The recovery rate of hirudin extracted by ethanol is low, and the activity of hirudin is lost. Although the recovery rate of hirudin extracted by acetone is higher, acetone itself is toxic, and the temperature needs to be strictly controlled during extraction to avoid inactivation of hirudin caused by high temperature. Enzymatic hydrolysis is a popular extraction method in recent years. Patent CN101332211A discloses a leech extract prepared by controllable enzymatic hydrolysis and a preparation method thereof. The leech extract is prepared by trypsin or / and pancreatic enzyme hydrolysis, organic solvent precipitation, ultrafiltration and anion exchange chromatography. In this technology, only trypsin or / and pancreatic enzyme is used, and the enzymatic hydrolysis efficiency is limited. Patent CN103251926A discloses a leech extract, a preparation method and application thereof. The leech is first hydrolyzed by pepsin under acidic conditions, and then the enzyme hydrolysis suspension is hydrolyzed by trypsin under alkaline conditions. The leech extract is then refined and purified by ultrafiltration, nanofiltration and cation exchange gel, and the purity of the leech extract is more than 90%. However, pepsin has a certain effect on hirudin, which can easily lead to inactivation. SUMMARY

[0004] To address the shortcomings of existing technologies, the first objective of this application is to provide a method for extracting hirudin, which involves extracting hirudin through freeze-drying, enzymatic hydrolysis, and filtration purification steps, resulting in high hirudin recovery rate and high activity.

[0005] The second objective of this application is to provide an oral formulation containing hirudin, wherein the enteric-coated tablet is coated with a coating material, exhibiting excellent sustained-release and anticoagulant effects, and can be used to treat and prevent chronic thrombotic diseases, thereby overcoming the shortcomings of oral polypeptide drugs.

[0006] The third objective of this application is to provide a method for preparing an oral formulation containing hirudin, which is simple and easy to operate.

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0008] According to one aspect of this application, a method for extracting hirudin is provided, comprising the following steps:

[0009] S1. Soak fresh leeches in a freeze-drying protectant, remove them 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;

[0010] S2. After pulverizing the freeze-dried leeches, add them to water, along with enzymes and lecithin. Enzymatically hydrolyze the leeches at 35–40°C and pH 7.5–8.5 for 4–6 hours, then cool to room temperature to obtain the enzymatic hydrolysis product.

[0011] S3. Sterilization, ultrafiltration, and drying yield hirudin.

[0012] Further, in step S1, the freeze-drying protectant, by weight percentage, comprises: 5-10% sucrose, 5-8% trehalose, and the remainder is water.

[0013] Furthermore, in step S1, the soaking is carried out at a temperature range of room temperature to 37°C for 2 to 4 hours. During the soaking process, the weight ratio of the freeze-drying protectant to the leeches is (10 to 20): 1.

[0014] This application involves immersing leeches in a freeze-drying protectant before freeze-drying. This protectant penetrates the leech tissue, facilitating the disruption of cell membranes and other structures during subsequent freezing, promoting the dissolution of hirudin, and increasing hirudin recovery. Furthermore, the protectant provides some protection during the subsequent freeze-drying process, preventing conformational damage to hirudin and thus preserving high activity of the resulting hirudin. This application limits the immersion time of leeches in the freeze-drying protectant. Insufficient immersion time prevents the protectant from penetrating the leech tissue, while excessive immersion time may cause the dissolution of other components, affecting hirudin extraction to some extent. Experiments have shown that prolonged immersion time reduces the hirudin recovery rate.

[0015] In this application, pre-freezing at -40℃ to -50℃, instead of direct freezing at -20℃ to -30℃, helps to increase the freezing rate and avoids the formation of large ice crystals inside the leech cells due to slow freezing, which would damage the natural conformation of hirudin and affect its activity. However, the pre-freezing conditions need to be controlled to avoid uneven distribution of ice crystals inside the cells, which would affect the extraction of hirudin.

[0016] Furthermore, in step S2, the pulverization is carried out using a low-temperature pulverization method well known in the art, such as low-temperature ultrafine pulverization or low-temperature grinding, to prevent the temperature from being too high during the grinding process and affecting the activity of hirudin; the particle size of the pulverized material is less than 150 mesh, preferably 150 to 200 mesh, which is beneficial to the extraction of hirudin.

[0017] Furthermore, in step S2, the weight ratio of freeze-dried leeches to water is (8-15):1.

[0018] Further, 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 for 1g of freeze-dried leeches; when both alkaline protease and trypsin are added at the same time, the amount of trypsin is not less than 100 U / g.

[0019] Furthermore, in step S2, the amount of lecithin added is 0.3 to 0.7% of the weight of the freeze-dried leeches.

[0020] This application utilizes an enzymatic hydrolysis method to extract hirudin, employing alkaline protease and trypsin for enzymatic hydrolysis. To ensure enzyme activity and avoid excessively high temperatures that could damage hirudin activity, the hydrolysis is performed at 35–40°C, satisfying both the hydrolysis temperature and maintaining high activity of the resulting hirudin. Furthermore, it was found that adding a certain amount of lecithin during the enzymatic hydrolysis process helps disrupt the leech cell membrane structure, allowing hirudin to dissolve and be released into the water more effectively, and reducing hirudin aggregation. This reduces hirudin loss during subsequent filtration and purification, thereby improving the hirudin recovery rate. However, experiments showed that excessive lecithin addition not only fails to improve the hirudin recovery rate but also easily leads to a decrease in hirudin activity. Therefore, this application limits the amount of lecithin used.

[0021] Furthermore, in step S3, the sterilization is carried out using sterilization methods well known in the art, as long as the sterilization temperature is kept below 40°C to avoid damaging the activity of hirudin. It is preferred to use ultraviolet irradiation for sterilization.

[0022] Further, the ultrafiltration step in step S3 includes: filtering the sterilized enzymatic hydrolysate sequentially through a 10kDa to 30kDa ultrafiltration membrane and a 1000Da ultrafiltration membrane, and collecting the unfiltered liquid.

[0023] This application first uses an ultrafiltration membrane with a molecular weight cutoff of 10kDa to 30kDa to filter the sterilized enzymatic hydrolysate, retaining the enzyme and large molecular weight impurities such as proteins and peptides. Then, the permeate is filtered using an ultrafiltration membrane with a molecular weight cutoff of 1000Da to remove small molecular weight impurities such as free amino acids with a molecular weight below 1000Da, thereby obtaining hirudin.

[0024] Furthermore, the drying in step S3 is either spray drying or freeze drying. In this application, spray drying requires controlling the temperature below 40°C to avoid damaging the activity of hirudin.

[0025] According to another aspect of this application, an oral formulation containing hirudin is provided, comprising a core layer and a coating layer;

[0026] The core layer contains hirudin obtained by the extraction method described above, as well as pharmaceutically acceptable excipients;

[0027] The coating layer comprises the following raw materials in parts by weight: 1-3 parts carboxymethyl chitosan, 0.3-0.8 parts sorbitol, and 0.1-0.2 parts glyceryl monostearate.

[0028] Furthermore, the weight ratio of the core layer to the coating layer is 100:(4-8).

[0029] Furthermore, the hirudin content in the core layer is ≥50%, for example, it can be 50%, 60%, 70%, 80%, 90%, etc., including but not limited to any one or more combinations of diluents, excipients, fillers, binders, humectants, and absorption promoters. It should be noted that the diluents, excipients, fillers, binders, humectants, and absorption promoters listed above are all commonly used excipients in the art, and therefore will not be described in detail here. Specifically, in the following specific embodiments section, starch and microcrystalline cellulose are added as excipients.

[0030] Furthermore, the degree of carboxymethyl substitution (DS) of the carboxymethyl chitosan is 0.3–0.45, and the molecular weight is 50 kDa–70 kDa.

[0031] Furthermore, the oral formulation is a tablet or capsule.

[0032] Hirudin, when administered orally, is easily degraded and enzymatically hydrolyzed in the gastrointestinal tract, resulting in low bioavailability. However, when administered via injection, its efficacy is short-lived, and frequent injections cause significant physical and psychological distress for patients requiring long-term anticoagulation. Current methods involve coating hirudin with a material to resist gastric acid degradation, prolonging disintegration time and allowing for absorption in the colon.

[0033] This application uses carboxymethyl chitosan as the main coating material to encapsulate hirudin to prepare an oral formulation. By selecting carboxymethyl chitosan with specific dissipation density (DS) and molecular weight, it is made soluble only in an environment with pH > 6.5, i.e., in the colon, avoiding premature dissolution in the small intestine, thus improving colonic targeting. Furthermore, it can be rapidly degraded by the colonic flora, resulting in rapid disintegration and enabling rapid drug delivery. The resulting oral formulation also exhibits good high-temperature stability, overcoming the problem of hirudin's activity being easily destroyed at high temperatures. While increasing the DS and molecular weight of carboxymethyl chitosan improves its film-forming properties, excessively high carboxymethyl content can lead to increased solubility in the intestinal environment, affecting colonic targeting and the function of colonic flora, slowing down colonic degradation, and also affecting its high-temperature stability. Conversely, excessively high molecular weight can also affect degradation and drug release. Conversely, decreasing the molecular weight of carboxymethyl chitosan leads to poor film-forming properties, easy film breakage, and affects targeted drug release and stability. This application adds a certain amount of sorbitol and glyceryl monostearate to the outer coating layer, which can increase film-forming properties, prevent the outer coating layer from breaking, thereby improving targeted drug release and high-temperature stability. In addition, the lecithin retained in hirudin also helps the coating material spread, making the film layer more uniform, which helps to improve targeted drug release and high-temperature stability.

[0034] According to another aspect of this application, a method for preparing an oral formulation containing hirudin is provided, comprising the following steps:

[0035] The core layer was prepared by mixing hirudin with pharmaceutically acceptable excipients. A coating solution was prepared by adding 1-3 parts of carboxymethyl chitosan, 0.3-0.8 parts of sorbitol and 0.1-0.2 parts of glyceryl monostearate to 100 parts of water. The coating layer was then coated on the surface of the core layer by spray coating method, resulting in a core layer weight gain of 3-7%.

[0036] Furthermore, in the spray coating method, the coating liquid is placed in a fluidized bed coating machine, the inlet air temperature is controlled to be no higher than 40℃, the outlet air temperature is controlled to be no higher than 35℃, the spray speed is 10-18 rpm, and the spray pressure is adjusted to 0.2-0.3 MPa.

[0037] Furthermore, when the oral formulation is a capsule, it also includes filling the capsule shell at the end.

[0038] Compared with the prior art, this application has the following beneficial effects:

[0039] 1. This application provides a method for extracting hirudin, including freeze-drying, enzymatic hydrolysis, and filtration purification steps. By soaking the hirudin in a freeze-drying protectant before freeze-drying and adding lecithin during enzymatic hydrolysis, the recovery rate and activity of hirudin are high.

[0040] 2. The leech micro powder provided in this application is prepared by encapsulating hirudin with carboxymethyl chitosan. It disintegrates slowly in the stomach and small intestine but quickly in the colon, which improves colon targeting and has high bioavailability. Detailed Implementation

[0041] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of this application, but do not limit this application in any way. The following content is merely an exemplary description of the scope of protection claimed in this application, and those skilled in the art can make various changes and modifications to the invention based on the disclosed content, which should also fall within the scope of protection claimed in this application.

[0042] The present application will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of this application are obtained through conventional commercial means.

[0043] Example 1

[0044] This embodiment provides a method for extracting hirudin, including the following steps:

[0045] S1. Place fresh leeches in 20 times their weight of freeze-drying protectant and soak them at room temperature for 4 hours. After removing them, wipe the surface dry and then pre-freeze them at -40℃ for 2 hours. Then freeze-dry them at -20℃ for 6 hours to obtain freeze-dried leeches.

[0046] The freeze-drying protectant contains: 5% sucrose, 8% trehalose, and the remainder is water;

[0047] S2. After crushing the freeze-dried leeches through a 150-mesh sieve, add them to water. Add 200U of trypsin and 0.003g of lecithin to every 1g of freeze-dried leeches. Enzymatically hydrolyze for 6 hours at 35℃ and pH=8.5. Cool to room temperature to obtain crude extract.

[0048] 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 unpermeated liquid to obtain the purified hirudin solution, and finally spray dry to obtain hirudin.

[0049] Example 2

[0050] This embodiment provides another method for extracting hirudin, including the following steps:

[0051] S1. Place fresh leeches in 20 times their weight of freeze-drying protectant, soak at 37°C for 2 hours, remove 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.

[0052] The freeze-drying protectant contains: 10% sucrose, 5% trehalose, and the remainder is water;

[0053] S2. After crushing the freeze-dried leeches through a 200-mesh sieve, add them to water. Add 500U of alkaline protease and 0.007g of lecithin to every 1g of freeze-dried leeches. Enzymatically hydrolyze for 6 hours at 40℃ and pH=7.5. Cool to room temperature to obtain crude extract.

[0054] 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 unpermeated liquid to obtain the purified hirudin solution, and finally spray dry to obtain hirudin.

[0055] Example 3

[0056] This embodiment provides another method for extracting hirudin. The difference from Embodiment 2 is that in step S1, the fresh leeches are soaked in the freeze-drying protectant for 6 hours; the rest is the same as in Embodiment 2.

[0057] Example 4

[0058] This embodiment provides another method for extracting hirudin. The difference from Embodiment 2 is that in step S1, the fresh leeches are not soaked in the freeze-drying protectant, but are directly mixed with the freeze-drying protectant and then pre-frozen; the rest is the same as in Embodiment 2.

[0059] Example 5

[0060] This embodiment provides another method for extracting hirudin, which differs from Embodiment 2 in that the fresh leeches are not soaked in the freeze-drying protectant in step S1; the rest is the same as in Embodiment 2.

[0061] Example 6

[0062] This embodiment provides another method for extracting hirudin, which differs from Embodiment 2 in that the pre-freezing temperature in step S1 is -60℃; the rest is the same as in Embodiment 2.

[0063] Example 7

[0064] This embodiment 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 in Example 2.

[0065] Example 8

[0066] This embodiment provides another method for extracting hirudin, which differs from Example 2 in that 0.012g of lecithin is added during the enzymatic hydrolysis process in step S2; the rest is the same as in Example 2.

[0067] Example 9

[0068] This embodiment provides another method for extracting hirudin, which differs from Embodiment 2 in that lecithin is not added during the enzymatic hydrolysis process in step S2; the rest is the same as in Embodiment 2.

[0069] Example 10

[0070] This embodiment provides another method for extracting hirudin, which differs from Embodiment 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 Embodiment 2.

[0071] Experimental Example 1

[0072] This experiment used the thrombin titration method to test the recovery rate and activity of hirudin obtained according to the methods provided in Examples 1-10 above. The principle is based on the fact that the binding ratio of hirudin to thrombin is 1:1 (mol / mol); the international unit for thrombin is NIH, and the activity of hirudin is expressed in antithrombin activity units (ATU), where one AUT equals the amount of hirudin neutralized in one NIH thrombin. Specifically, the activity A1 of hirudin in the crude extract obtained in step S2 and the activity A2 of hirudin in the purified hirudin solution obtained in step S3 (i.e., the activity of the product hirudin) were tested, and the recovery rate of hirudin was calculated according to the formula: Recovery rate = A2 / A1 × 100%.

[0073] The test results are shown in Table 1 below. It can be seen that the hirudin obtained by the extraction method provided in this application has high activity and high recovery rate.

[0074] Table 1

[0075]

[0076] Example 11

[0077] This embodiment provides an oral formulation containing leech powder, comprising a core layer and a coating layer;

[0078] The core layer consists of hirudin obtained in Examples 1-10 above and pharmaceutically acceptable excipients;

[0079] The coating layer comprises the following raw materials in parts by weight: 1-3 parts carboxymethyl chitosan, 0.3-0.8 parts sorbitol, and 0.1-0.2 parts glyceryl monostearate;

[0080] Among them, the degree of carboxymethyl substitution (DS) of carboxymethyl chitosan is 0.3 to 0.45, and the molecular weight is 50 kDa to 70 kDa.

[0081] The preparation method of the above-mentioned leech powder includes the following steps:

[0082] The core layer was prepared by mixing hirudin with pharmaceutically acceptable excipients. A coating solution was prepared by adding 1-3 parts of carboxymethyl chitosan, 0.3-0.8 parts of sorbitol and 0.1-0.2 parts of glyceryl monostearate to 100 parts of water. The coating layer was then coated on the surface of the core layer by spray coating method, resulting in a core layer weight gain of 3-7%.

[0083] The key to this oral formulation with colon-targeted drug release lies in the outer coating. Therefore, to verify the colon-targeting nature of the oral formulation of this application, this application prepared tablets for testing with the following example: the weight ratio of hirudin in the core layer to a pharmaceutically acceptable carrier (fixed at a weight ratio of 2:1 microcrystalline cellulose and starch) was 7:3, and the core layer weight gain was 3%.

[0084] The following tablets were prepared according to the above formula and method, as detailed below:

[0085] Tablets 1# to 10#

[0086] The hirudin in the core layer is hirudin obtained in Examples 1 to 10 above;

[0087] The coating layer contains the following raw materials in parts by weight: 3 parts carboxymethyl chitosan (DS=0.3, molecular weight 70kDa), 0.3 parts sorbitol, and 0.1 parts glyceryl monostearate.

[0088] The hirudin obtained in Example 2 was used, and the coating material formulation was the same as that of tablets 1# to 8#.

[0089] Tablets 11#~16#

[0090] Tablets 11# to 16# use the hirudin obtained in Example 2, and the specific coating layer formulation is shown in Table 2 below.

[0091] Table 2

[0092]

[0093] Experimental Example 2

[0094] The in vitro release performance of the tablets prepared in Example 11 was tested. Specifically, the tablets were first placed in 0.1 mol / L hydrochloric acid solution (simulating artificial gastric fluid) and samples were taken for measurement after 4 hours; then transferred to phosphate buffer solution at pH 6.5 (simulating artificial intestinal fluid) and samples were taken for measurement after 6 hours; finally transferred to phosphate buffer solution at pH 7.5 (simulating artificial colon fluid) and samples were taken for measurement after 7 hours and 8 hours. The cumulative release percentage was calculated, noting that all the above times are cumulative times.

[0095] The test results are shown in Table 3 below. It can be seen that the oral formulation containing hirudin provided in this application has excellent colon-targeted drug release properties. Although there is some drug release in the gastric and intestinal fluid environments at pH 1.2 and pH 6.5, the release amount is very small and negligible. However, at pH 7.5, the release amount reaches 90% within 2 hours, indicating rapid release in the colonic environment and good colon-targeted drug release properties. Comparing tablet #2 and tablets #12-16, 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 work together to give the coating good colon-targeted drug release properties. In addition, the addition of sorbitol and glyceryl monostearate to the coating also helps to improve its disintegration performance.

[0096] Table 3

[0097]

[0098] Experimental Example 3

[0099] The stability of tablets #2, #8~10, and #12~16 prepared in Example 11 was tested. Following the requirements of the influencing factor testing principles in Appendix XIX C of the 2010 edition of the Chinese Pharmacopoeia, Part II, the tablets were placed in petri dishes and incubated at 40℃ and 60℃, respectively, and samples were taken for analysis after 5 and 10 days. The results are shown in Table 4 below. It can be seen that the oral formulations prepared by the method described in this application have good stability and maintain high antithrombin activity even under high-temperature conditions.

[0100] Table 4

[0101]

[0102] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, this application is not limited to the above embodiments, and 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 protection scope of this application.

Claims

1. A method for extracting hirudin, characterized in that, Includes the following steps: S1. Soak fresh leeches in a freeze-drying protectant, remove them 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; The freeze-drying protectant, by weight percentage, comprises: 5-10% sucrose, 5-8% trehalose, and the remainder is water; S2. Pulverize the freeze-dried leeches and add them to water. Add enzymes and lecithin, and enzymatically hydrolyze at 35-40℃ and pH=7.5-8.5 for 4-6 hours. Cool to room temperature to obtain the enzymatic hydrolysis product. The amount of lecithin added is 0.3-0.7% of the weight of the freeze-dried leeches. S3. Sterilization, ultrafiltration, and drying yield hirudin.

2. The extraction method according to claim 1, characterized in that, In step S1, the soaking is carried out in the temperature range of room temperature to 37°C, and the soaking time is 2 to 4 hours.

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

4. The extraction method according to claim 1, characterized in that, The ultrafiltration step in step S3 includes: filtering the sterilized enzymatic hydrolysate sequentially through a 10kDa to 30kDa ultrafiltration membrane and a 1000Da ultrafiltration membrane, and collecting the unfiltered liquid.

5. An oral preparation containing hirudin, characterized in that, It includes a core layer and a coating layer; The core layer comprises hirudin obtained by the extraction method according to any one of claims 1-4 and pharmaceutically acceptable excipients; The coating layer comprises the following raw materials in parts by weight: 1-3 parts of carboxymethyl chitosan, 0.3-0.8 parts of sorbitol, and 0.1-0.2 parts of glyceryl monostearate; the degree of carboxymethyl substitution (DS) of the carboxymethyl chitosan is 0.3-0.45, and the molecular weight is 50-70 kDa; The weight ratio of the core layer to the coating layer is 100:(4-8).

6. The oral preparation containing hirudin according to claim 5, characterized in that, The core layer contains ≥50% hirudin.

7. A method for preparing the oral formulation containing hirudin as described in claim 5 or 6, characterized in that, Includes the following steps: The core layer was prepared by mixing hirudin with pharmaceutically acceptable excipients. A coating solution was prepared by adding 1-3 parts of carboxymethyl chitosan, 0.3-0.8 parts of sorbitol and 0.1-0.2 parts of glyceryl monostearate to 100 parts of water. The coating layer was then coated on the surface of the core layer by spray coating method, resulting in a core layer weight gain of 3-7%.

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