Leech extract as well as preparation method and application thereof

Abstract

The invention discloses a leech extract and a preparation method and application thereof, and relates to the technical field of leech extract preparation, the leech extract comprises leech oil, hirudin, mixed polypeptide and a small molecule extract, the small molecule extract is a small molecule compound and trace elements, the small molecule compounds are small molecule oligopeptides, pyridines, glycolipids, carboxylic esters and steroids, and the trace elements comprise Fe, Mn, Zn and Co; according to the leech extract and the preparation method and application thereof, through low-temperature, high-pressure and organic solvent subcritical efficient extraction, living leeches are instantly killed, biological enzymes are inactivated, leech oil in the leeches is effectively extracted, peculiar smells are removed, meanwhile, thermosensitive functional components are protected, and the extraction efficiency of hirudin is improved; ultrasonic extraction is performed through a continuous countercurrent ultrasonic extractor, so that the leaching efficiency of the hirudin is improved; neutral or alkaline protease is used for enzymolysis, protein is converted into high-activity oligopeptide, the utilization rate of effective components is increased, and the peculiar smell of the product is reduced.

Description

Technical Field

[0001] The invention relates to a leech extract preparation technology, and in particular to a leech extract and a preparation method and application thereof. Background Art

[0002] Leeches are commonly used traditional Chinese medicines for promoting blood circulation and removing blood stasis. In recent years, leeches and their extracts have been widely used in contemporary medical fields such as cardiovascular disease, microsurgery, tumors, diabetes and its complications, infectious diseases, arthritis and pain. The extraction methods of leech extracts are mainly divided into two categories, traditional extraction methods (water extraction, salt precipitation, organic solvent extraction) and bionic induction methods. In addition, in recent years, ultrasound and enzymatic hydrolysis have also been used to try to extract leech extracts.

[0003] In the prior art process, when extracting leech extract, water is used for low-temperature extraction, and the yield is low; and when separating the leech extract, proteins larger than 10,000 Da and some active proteins are separated and removed, so that the proteins and some active proteins are not fully utilized, which not only causes a waste of resources, but also may reduce the overall value and utility of the leech extract. Summary of the invention

[0004] The purpose of the present invention is to provide a leech extract and a preparation method and application thereof, so as to solve the above-mentioned deficiencies in the prior art.

[0005] In order to achieve the above object, the present invention provides the following technical solution: a leech extract, comprising leech oil, hirudin, mixed polypeptides and small molecule extracts.

[0006] Furthermore, the small molecule extracts are small molecule compounds and trace elements.

[0007] Furthermore, the small molecule compounds are small molecule oligopeptides, pyridines, glycolipids, carboxylates and steroids, and the trace elements include Fe, Mn, Zn and Co.

[0008] A method for preparing a leech extract, comprising:

[0009] S1, taking 500 kg of living leeches and putting them into a subcritical ultrasonic extraction tank, and extracting them by subcritical ultrasonic method, thereby obtaining leech oil;

[0010] S2. Put the de-oiled leeches into a 3m 3 In an ultrasonic extraction tank, 6 to 8 times amount of pure water is added to perform ultrasonic extraction, and then separated by a 500 kg / h horizontal screw centrifuge and then separated by a 500 kg / h butterfly centrifuge to obtain a supernatant and a precipitate; the supernatant is filtered and separated by a 10000 Da ultrafiltration membrane to obtain a filtrate 1 and a filtrate 1;

[0011] S3, put the filtrate into 3m 3 In the enzymatic hydrolysis tank, add 1% to 3% of the volume of the filtrate into the neutral or alkaline protease and hydrolyze for 2 to 4 hours. The filtrate after hydrolysis is centrifuged by butterfly centrifuge, and the clear liquid is concentrated by reverse osmosis membrane, 500kg / h low temperature external circulation concentrator, 50m 2 After drying in a vacuum low-temperature drying unit, a mixed polypeptide product is obtained;

[0012] S4, filtering the filtrate 1 with a 5000Da ultrafiltration membrane to obtain a filtrate 2 and a filtrate 2; preparing hirudin from the filtrate 2, filtering the filtrate 2 with a nanofiltration membrane to remove salt, and then concentrating it with a reverse osmosis membrane, and concentrating it with a 500kg / h low-temperature external circulation concentrator, 50m 2 After drying in a vacuum low-temperature drying unit, a small molecule extract product is prepared;

[0013] S5. Leech extract is obtained by combining leech oil, hirudin, mixed polypeptides and small molecule extracts.

[0014] Furthermore, the extraction time of the subcritical ultrasonic extraction in S1 is 30 minutes.

[0015] Furthermore, the ultrasonic extraction conditions in S2 are: ultrasonic temperature 35-55°C, and ultrasonic time 20-40 min.

[0016] Further, the filtrate 2 is used to prepare hirudin in S4, and the preparation method is: the filtrate 2 is concentrated by reverse osmosis membrane, concentrated by 500kg / h low temperature external circulation concentrator, 50m 2 The product is dried by a vacuum low-temperature drying unit to obtain hirudin.

[0017] Further, the filtrate 2 is used to prepare hirudin in S4, and the preparation method is as follows: the filtrate 2 is separated by a preparative dextran gel separation column, the eluate is concentrated by a reverse osmosis membrane, and concentrated by a 500 kg / h low-temperature external circulation concentrator, and 50 m 2 After drying in a vacuum low-temperature drying unit, the hirudin product is obtained.

[0018] The invention discloses an application of a leech extract in preparing cardiovascular and cerebrovascular disease-related medicines and health products.

[0019] The invention discloses an application of the leech extract prepared by the preparation method in the preparation of cardiovascular and cerebrovascular disease related medicines and health products.

[0020] Compared with the prior art, the leech extract provided by the present invention and its preparation method and application adopt subcritical ultrasonic extraction technology, and through low temperature, high pressure and organic solvent subcritical high-efficiency extraction, the living leeches are instantly killed and the biological enzymes are inactivated, the leech oil in the leeches is effectively extracted and the odor is removed, while the heat-sensitive functional components are protected and the extraction efficiency of hirudin is improved;

[0021] The de-oiled leeches are ultrasonically extracted by a continuous countercurrent ultrasonic extractor, and the cell membrane is broken by cavitation effect and mechanical action, which accelerates the movement of solvent molecules and improves the efficiency of leech extraction. Neutral or alkaline proteases are used for enzymatic hydrolysis to convert proteins into highly active oligopeptides, thereby improving the utilization rate of effective ingredients and reducing product odor.

[0022] The preparation method of the leech extract is large-scale and automated. By adopting advanced equipment to form a production line, efficient, energy-saving and environmentally friendly production is achieved, so that the leech raw materials are fully utilized, production efficiency is improved, costs are reduced, and the market competitiveness of the enterprise is enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0024] Figure 1 A schematic flow chart of the overall preparation method provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0026] Embodiment 1:

[0027] A leech extract comprises leech oil, hirudin, mixed polypeptides and small molecule extracts; the small molecule extracts are small molecule compounds and trace elements; the small molecule compounds are small molecule oligopeptides, pyridines, glycolipids, carboxylates and steroids, and the trace elements include Fe, Mn, Zn and Co.

[0028] The specific implementation method is that leeches are commonly used traditional Chinese medicines for promoting blood circulation and removing blood stasis. In recent years, leeches and their extracts have been widely used in contemporary medical fields such as cardiovascular diseases, microsurgery, tumors, diabetes and its complications, infectious diseases, arthritis and pain. Leeches are oligochaete, hermaphrodites, and higher invertebrates. The leeches included in the 2020 edition of the Pharmacopoeia of the People's Republic of my country are the dried whole bodies of leeches, leeches or willow leaf leeches of the Hirudinidae family. They are caught in summer and autumn, scalded to death with boiling water, and dried in the sun or at low temperature. The main components of leeches are protein and polypeptide macromolecules, which can be divided into two categories according to their pharmacological effects. One category is the active ingredients that directly act on the coagulation system, such as the well-known hirudin, heparinoids, hirudin, histamine, etc.; the other category is other protease inhibitor components, such as thrombin inhibitors, etc. In addition, leeches also contain some small molecule compounds and trace elements. Small molecule compounds include pyridines, glycolipids, carboxylates and steroids, and trace elements include Fe, Mn, Zn, Co, etc.

[0029] Leech oil has the effects and functions of promoting blood circulation, removing blood stasis, reducing swelling and relieving pain, improving blood circulation, promoting wound healing, and regulating blood lipids. Leech oil can effectively promote blood circulation and relieve pain and discomfort caused by blood stasis; for symptoms such as trauma and swelling, leech oil can reduce local swelling and relieve pain; leech oil helps prevent and improve vascular-related diseases by improving blood flow in blood vessels; leech oil contains ingredients that promote tissue repair and can accelerate the wound healing process; leech oil can help regulate blood lipids and is beneficial to maintaining cardiovascular health.

[0030] Hirudin is a protein in leeches, containing 65 amino acid residues and 3 pairs of disulfide bonds. It can directly prevent the binding of thrombin to fibrin without the help of cofactors. Because it has no immune response to red blood cells, it can be used to treat platelet abnormalities and heparin-induced thrombocytopenia. Many studies have shown that hirudin is more effective than heparin in treating deep vein thrombosis and patients with local ischemic diseases such as unstable angina. Unlike heparin, hirudin can prevent the occurrence of high-risk cardiovascular diseases, treat intravascular coagulation syndrome, and relieve pulmonary embolism and venous thrombosis.

[0031] Embodiment 2:

[0032] This embodiment provides a technical solution based on the first embodiment: a method for preparing a leech extract, comprising:

[0033] S1, taking 500 kg of living leeches and putting them into a subcritical ultrasonic extraction tank, and extracting them by subcritical ultrasonic extraction for 30 minutes, thereby obtaining leech oil;

[0034] The specific implementation method is that subcritical ultrasonic extraction is an extraction method that combines subcritical extraction (Sub-critical luid extraction technology) and ultrasound-assisted extraction technology. Subcritical extraction is a technology that uses solvent 1,1,1,2-tetrafluoroethane (R134a) in a subcritical state (the system pressure and temperature are controlled to keep the solvent 1,1,1,2-tetrafluoroethane (R134a) in a liquid state. At this time, the solvent 1,1,1,2-tetrafluoroethane (R134a) has liquid solubility and gaseous fluidity, which greatly increases the efficiency of leaching); ultrasound-assisted extraction technology increases the damage to cell walls through ultrasonic cavitation, thereby improving the extraction efficiency. The combination of ultrasonic treatment and subcritical extraction can effectively increase the dissolution rate, the extraction process is clean, and the extract can be effectively protected from being destroyed.

[0035] Subcritical ultrasonic extraction technology is used to extract the leech oil that needs to be removed from the leech, effectively removing the leech oil and odor that affect subsequent production for the extraction of hirudin, and at the same time obtaining high-nutrition, high-value-added leech oil. Subcritical ultrasonic extraction has the characteristics of low temperature, high pressure, and organic solvent subcritical high-efficiency extraction. While efficiently extracting leech oil, it instantly kills the living leech and inactivates the biological enzymes in the body, effectively protecting the heat-sensitive active ingredients in the leech, and effectively ensuring the extraction of subsequent active ingredients.

[0036] S2. Put the de-oiled leeches into a 3m 3 In an ultrasonic extraction tank, 6 to 8 times amount of pure water is added, and ultrasonic extraction is performed at 35 to 55° C. for 20 to 40 minutes, and then separated by a 500 kg / h horizontal screw centrifuge and then by a 500 kg / h butterfly centrifuge to obtain a supernatant and a precipitate; the supernatant is filtered and separated by a 10000 Da ultrafiltration membrane to obtain a filtrate 1 and a filtrate 1;

[0037] The specific implementation method is that in the 500kg / h horizontal screw centrifuge, 500kg / h refers to the processing capacity of the horizontal screw centrifuge, that is, the weight of the material that can be processed per hour is 500kg; in the 500kg / h butterfly centrifuge, 500kg / h refers to the processing capacity of the butterfly centrifuge, that is, the weight of the material that can be processed per hour is 500kg. The horizontal screw centrifuge is suitable for processing suspensions with larger solid phase particle size and larger solid-liquid density difference, and can efficiently perform solid-liquid separation. The butterfly centrifuge is usually used to process suspensions with smaller solid phase particle size and smaller solid-liquid density difference, especially in situations where higher liquid clarity is required. Using a horizontal screw centrifuge to process larger solid phase particles first, and then using a butterfly centrifuge to further process smaller solid phase particles can improve the overall separation efficiency.

[0038] An ultrasonic generator is used to emit ultrasonic waves into the extraction solvent. The "cavitation effect" and mechanical action generated by the ultrasonic waves in the extraction solvent can effectively break the leech cell membrane, so that the effective ingredients are in a free state and dissolved in the extraction solvent. On the other hand, it can accelerate the molecular movement of the extraction solvent, so that the effective ingredients in the extraction solvent and the leech can quickly contact, merge and mix with each other, which is beneficial to the leaching of hirudin. Ultrasonic low-temperature extraction also protects the heat-sensitive effective ingredients from being destroyed. Continuous countercurrent ultrasonic extractor extraction makes its production efficient, energy-saving, environmentally friendly and large-scale.

[0039] The precipitate generated in this step is collected and fermented to produce a feed additive to improve the utilization rate of leech raw materials. The precipitate is mixed with glycerol and then placed in an ultrasonic oscillator for ultrasonic oscillation to extract the effective components in the leech; the extracted reserve liquid (liquid containing the effective components of the leech) is placed in a container, an appropriate amount of vitamins is added, the pH value is adjusted to 5.0, and sterilization is performed to obtain a reserve liquid B; the reserve liquid B is placed in a fermentation tank filled with TB culture medium, lactose and other nutrients are added, and anaerobic fermentation is performed to obtain a fermentation liquid.

[0040] The fermentation liquid is filtered to remove solid impurities to obtain a clear fermentation liquid, which can be mixed with other feed additive ingredients, such as vitamins, mycelium, pyruvic acid, sodium sulfide, etc., to make a feed additive with microbial fermentation products. This feed additive can improve breeding efficiency, reduce breeding costs, and utilize the comprehensive effects of mycelium to reduce the cost superposition caused by multiple substances. The prepared feed additive is inspected to ensure that it meets the quality standards before packaging. It can be weighed and bagged during packaging to ensure that the product is easy to use and store.

[0041] Through the above steps, the leech sediment can be converted into a feed additive with high nutritional value and biological activity, providing an efficient, safe and sustainable feed source for the aquaculture industry.

[0042] S3, put the filtrate into 3m 3 In the enzymatic hydrolysis tank, add 1% to 3% of the volume of the filtrate into the neutral or alkaline protease and hydrolyze for 2 to 4 hours. The filtrate after hydrolysis is centrifuged by butterfly centrifuge, and the clear liquid is concentrated by reverse osmosis membrane, 500kg / h low temperature external circulation concentrator, 50m 2 After drying in a vacuum low-temperature drying unit, a mixed polypeptide product is obtained;

[0043] The specific implementation method is that the reverse osmosis technology is based on the pressure difference as the driving force, and the solvent (such as water) is separated from the solution through a semi-permeable membrane. When the pressure applied to the feed liquid side exceeds the osmotic pressure difference on both sides of the membrane, the solvent will reverse the natural osmosis direction and the solute will be intercepted by the reverse osmosis membrane; the reverse osmosis concentration process does not involve heating, no phase change, low energy consumption, can maintain the original properties of the substance to a large extent, improve the concentration efficiency and reduce energy consumption; the reverse osmosis membrane material includes cellulose acetate and polyamide membrane, and its salt retention rate is 94% to 97%.

[0044] By using scientifically screened neutral or alkaline proteases and combining the enzymatic hydrolysis process carefully determined by single factor and orthogonal experiments, proteins can be efficiently converted into highly active oligopeptides. This process not only significantly improves the utilization rate of active ingredients, but also effectively reduces the odor of the product, making the final product purer and easier to accept. These converted oligopeptides greatly improve the original functional properties of proteins and further enhance the effects of all functional elements in leeches, thereby broadening its application range in various fields.

[0045] During the processing, neutral proteases or alkaline proteases are used to hydrolyze macromolecular proteins with molecular weights greater than hirudin. The purpose of this is to decompose these macromolecular proteins that originally do not have specific physiological activities into smaller, more biologically active small-molecule oligopeptides. At the same time, this finely controlled enzymatic hydrolysis technology can also ensure that the hirudin in the extract will not be destroyed and thus be preserved intact. In this way, the effective utilization of raw materials is achieved, and the quality and quantity of key active ingredients are guaranteed, laying a solid foundation for subsequent product development.

[0046] S4, filtering the filtrate 1 with a 5000Da ultrafiltration membrane to obtain a filtrate 2 and a filtrate 2; preparing hirudin from the filtrate 2, filtering the filtrate 2 with a nanofiltration membrane to remove salt, and then concentrating it with a reverse osmosis membrane, and concentrating it with a 500kg / h low-temperature external circulation concentrator, 50m 2 After drying in a vacuum low-temperature drying unit, a small molecule extract product is prepared; the filtrate II is used to prepare hirudin, and the preparation method is as follows: the filtrate II is concentrated by a reverse osmosis membrane, concentrated by a 500kg / h low-temperature external circulation concentrator, and 50m 2 The product is dried by a vacuum low-temperature drying unit to obtain hirudin.

[0047] S5. Leech extract is obtained by combining leech oil, hirudin, mixed polypeptides and small molecule extracts.

[0048] The preparation method makes full use of leech raw materials, improves production efficiency, reduces production costs, and enables enterprises to have good competitive strength in the tide of market competition. At the same time, unused filtrate or filtrate residue is concentrated by a 500kg / h external circulation concentrator, the recovered water is reused, and the concentrated liquid is used to produce feed additives. Leech oil, hirudin, small molecule extracts and mixed polypeptides are obtained through comprehensive production, and they are sold separately or mixed in proportion according to market demand to ensure high cost-effective market sales.

[0049] Embodiment three:

[0050] This embodiment provides a technical solution based on the first embodiment: a method for preparing a leech extract, comprising:

[0051] S1, taking 500 kg of living leeches and putting them into a subcritical ultrasonic extraction tank, and extracting them by subcritical ultrasonic extraction for 30 minutes, thereby obtaining leech oil;

[0052] S2. Put the de-oiled leeches into a 3m 3 In an ultrasonic extraction tank, 6 to 8 times amount of pure water is added, and ultrasonic extraction is performed at 35 to 55° C. for 20 to 40 minutes, and then separated by a 500 kg / h horizontal screw centrifuge and then by a 500 kg / h butterfly centrifuge to obtain a supernatant and a precipitate; the supernatant is filtered and separated by a 10000 Da ultrafiltration membrane to obtain a filtrate 1 and a filtrate 1;

[0053] S3, put the filtrate into 3m 3 In the enzymatic hydrolysis tank, add 1% to 3% of the volume of the filtrate into the neutral or alkaline protease and hydrolyze for 2 to 4 hours. The filtrate after hydrolysis is centrifuged by butterfly centrifuge, and the clear liquid is concentrated by reverse osmosis membrane, 500kg / h low temperature external circulation concentrator, 50m 2 After drying in a vacuum low-temperature drying unit, a mixed polypeptide product is obtained;

[0054] S4, filtering the filtrate 1 with a 5000Da ultrafiltration membrane to obtain a filtrate 2 and a filtrate 2; preparing hirudin from the filtrate 2, filtering the filtrate 2 with a nanofiltration membrane to remove salt, and then concentrating it with a reverse osmosis membrane, and concentrating it with a 500kg / h low-temperature external circulation concentrator, 50m 2 After drying in a vacuum low-temperature drying unit, a small molecule extract product is prepared; the filtrate II is used to prepare hirudin, and the preparation method is as follows: the filtrate II is separated by a preparative dextran gel separation column, the eluate is concentrated by a reverse osmosis membrane, and concentrated by a 500kg / h low-temperature external circulation concentrator. 2 After drying in a vacuum low-temperature drying unit, the hirudin product is obtained.

[0055] S5. Leech extract is obtained by combining leech oil, hirudin, mixed polypeptides and small molecule extracts.

[0056] Sephadex gel chromatography is a technique for separation based on molecular size. During the chromatography process, substances with different molecular weights move at different speeds in the gel column. Substances with larger molecular weights cannot enter the interior of the gel particles, so they flow along the gaps between the gel particles and flow out of the chromatography column first; while substances with smaller molecular weights can enter the mesh of the gel particles, encounter greater resistance, and therefore flow out last. This separation mechanism enables the Sephadex gel separation column to effectively separate different components in the filtrate 2 according to molecular weight to obtain eluent and effluent.

[0057] Through the separation of the dextran gel separation column, impurities with a size similar to that of the target molecule in the filtrate 2 can be removed, thereby improving the purity of the target molecule. The eluate and effluent obtained by the dextran gel separation column can be used for subsequent different process treatments. The eluate can be concentrated by a reverse osmosis membrane and vacuum low-temperature dried to obtain 98% of the hirudin. This separation provides a purer and clearer raw material for the subsequent process, which helps to improve the quality and yield of the final product.

[0058] The use of dextran gel separation columns can reduce the amount of materials that need to be processed in subsequent processes, especially when the molecular weight of the target molecule is close to that of the impurity molecule, which can significantly reduce the workload and cost in subsequent steps and improve the efficiency and economy of the entire production process.

[0059] In summary, the preparative dextran gel separation column plays a key role in separation and purification in this process, providing high-quality raw materials for the subsequent preparation of high-content hirudin and small molecule extracts; the leech oil that needs to be removed from the leech is extracted by subcritical ultrasonic extraction technology, and high-nutrition, high-value-added leech oil is obtained. At the same time, the living leeches are instantly killed to inactivate the biological enzymes in the body, effectively protecting the thermosensitive effective components in the leeches, so that they can be effectively retained in the subsequent extraction; the de-oiled leeches are efficiently extracted from the hirudin by a continuous countercurrent ultrasonic extractor, and the continuous countercurrent ultrasonic extractor extraction makes its production efficient, energy-saving, environmentally friendly and large-scale; neutral or alkaline proteases are used to convert proteins into highly active oligopeptides, the utilization rate of effective ingredients is improved, and the odor of the product is effectively reduced.

[0060] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A leech extract, characterized in that Including leech oil, hirudin, mixed peptides and small molecule extracts.

2. The leech extract according to claim 1, characterized in that The small molecule extracts are small molecule compounds and trace elements.

3. The leech extract according to claim 2, characterized in that: The small molecule compounds include small molecule oligopeptides, pyridines, glycolipids, carboxylates and steroids, and the trace elements include Fe, Mn, Zn and Co.

4. A method for preparing a leech extract, characterized in that: include: S1, taking 500 kg of living leeches and putting them into a subcritical ultrasonic extraction tank, and extracting them by subcritical ultrasonic method, thereby obtaining leech oil; S2. Put the de-oiled leeches into a 3m 3 In an ultrasonic extraction tank, 6 to 8 times amount of pure water is added to perform ultrasonic extraction, and then separated by a 500 kg / h horizontal screw centrifuge and then separated by a 500 kg / h butterfly centrifuge to obtain a supernatant and a precipitate; the supernatant is filtered and separated by a 10000 Da ultrafiltration membrane to obtain a filtrate 1 and a filtrate 1; S3, put the filtrate into 3m 3 In the enzymatic hydrolysis tank, add 1% to 3% of the volume of the filtrate into the neutral or alkaline protease and hydrolyze for 2 to 4 hours. The filtrate after hydrolysis is centrifuged by butterfly centrifuge, and the clear liquid is concentrated by reverse osmosis membrane, 500kg / h low temperature external circulation concentrator, 50m 2 After drying in a vacuum low-temperature drying unit, a mixed polypeptide product is obtained; S4, filtering the filtrate 1 with a 5000Da ultrafiltration membrane to obtain a filtrate 2 and a filtrate 2; preparing hirudin from the filtrate 2, filtering the filtrate 2 with a nanofiltration membrane to remove salt, and then concentrating it with a reverse osmosis membrane, and concentrating it with a 500kg / h low-temperature external circulation concentrator, 50m 2 After drying in a vacuum low-temperature drying unit, a small molecule extract product is prepared; S5. Leech extract is obtained by combining leech oil, hirudin, mixed polypeptides and small molecule extracts.

5. The method for preparing a leech extract according to claim 4, characterized in that: The extraction time of the subcritical ultrasonic extraction in S1 is 30 min.

6. The method for preparing a leech extract according to claim 4, characterized in that: The ultrasonic extraction conditions in S2 are: ultrasonic temperature 35-55°C, and ultrasonic time 20-40 min.

7. The method for preparing a leech extract according to claim 4, characterized in that: S4 is described in which the filtrate 2 is used to prepare hirudin, and the preparation method is as follows: the filtrate 2 is concentrated by reverse osmosis membrane, concentrated by 500kg / h low temperature external circulation concentrator, 50m 2 The product is dried by a vacuum low-temperature drying unit to obtain hirudin.

8. The method for preparing a leech extract according to claim 4, characterized in that: S4 is described in which the filtrate 2 is used to prepare hirudin, and the preparation method is as follows: the filtrate 2 is separated by a preparative dextran gel separation column, the eluate is concentrated by a reverse osmosis membrane, and concentrated by a 500 kg / h low-temperature external circulation concentrator. 2 After drying in a vacuum low-temperature drying unit, the hirudin product is obtained.

9. Use of the leech extract according to any one of claims 1 to 3 in the preparation of cardiovascular and cerebrovascular disease related medicines and health products.

10. Use of the leech extract prepared by the preparation method according to any one of claims 4 to 8 in the preparation of cardiovascular and cerebrovascular disease related drugs and health products.

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