A lumbricus extract, composition, preparation, preparation method and application

Abstract

This invention provides an earthworm extract, its preparation method, composition, pharmaceutical formulation, and application, belonging to the field of composition technology. The earthworm extract comprises component A with a molecular weight <10 kDa and component B with a molecular weight of 10-66 kDa. Qualitative and quantitative detection methods for the earthworm extract reveal that it contains aprotinin and lumbrokinase. This invention is the first to detect aprotinin in earthworms. This invention also provides a method for preparing the earthworm extract, which significantly improves the extraction rate, enzyme activity, and anticoagulant activity of the active ingredients.

Description

Technical Field

[0001] This invention belongs to the field of composition technology, specifically relating to an earthworm extract, its preparation method and application. Background Technology

[0002] Earthworms, also known as "dilong" in Traditional Chinese Medicine, are used as a medicinal herb with antipyretic, calming, diuretic, and antihypertensive effects. Earthworms are rich in protein, containing all ten essential amino acids required by the human body, with high content and nutritional value, even exceeding that of soybean protein. They are also high in unsaturated fatty acids, especially linoleic acid, which has anti-cancer, blood pressure-lowering, and anti-atherosclerotic effects. Furthermore, they are rich in vitamins and trace elements, making the medicinal value of earthworms a hot topic of research. As a representative animal-derived medicinal source, they contain abundant protein polypeptides, such as lumbrokinase, plasminogen activator, and collagenase, as well as various amino acids, and are attracting attention for their multiple effects including anti-thrombosis, thrombolysis, anticoagulation, and fibrinolysis. The Chinese Pharmacopoeia does not record identification methods for key components in earthworms. According to existing literature, the main active ingredient in earthworms is lumbrokinase (also known as earthworm fibrinolytic enzyme), a proteolytic enzyme with significant fibrinolytic and antithrombotic activities. Since its first isolation from earthworms in 1983, its clinical application value has become increasingly prominent. This enzyme has a molecular weight between 16 and 45 kDa and possesses a unique dual-directional thrombolytic mechanism: it can directly degrade fibrin and activate plasminogen activator, thereby efficiently dissolving microthrombi, improving microcirculation, repairing vascular endothelial cells, and inhibiting platelet aggregation and thrombus reformation. It is currently widely used in the prevention and treatment of cardiovascular and cerebrovascular diseases, endocrine and respiratory diseases.

[0003] There have been reports on ingredients containing earthworms, for example: The master's thesis, "Extraction, Separation, Purification and External Preparation Study of Earthworm Polypeptides," by Liu Ziwei, published on June 1, 2024, by Shandong University of Traditional Chinese Medicine, discloses the following method: Dried earthworms were pulverized and passed through a No. 5 sieve to obtain earthworm powder. 2 g of the powder was weighed, soaked in 20 mL of distilled water for 1 h, and then extracted by reflux for 1 h. After cooling to room temperature, the mixture was centrifuged at 5000 r·min⁻¹ for 15 min to obtain the supernatant. The precipitate was washed twice with 3 mL of distilled water and centrifuged again at 5000 r·min⁻¹ for 15 min. The supernatants were combined, and 60 mL of anhydrous ethanol was added to the supernatant. After standing at room temperature for 24 h, the ethanol was evaporated in a 50℃ water bath. The mixture was then frozen at -20℃ for 24 h and freeze-dried to obtain lyophilized powder.

[0004] For example, Chinese patent CN1679681A discloses a traditional Chinese medicine preparation for treating Alzheimer's disease and its preparation method, which involves the preparation process of earthworm. However, in actual production, it has been found that the earthworm extraction process has a significant impact on the activity and yield of the earthworm extract. The enzyme activity, purity, and yield of the earthworm extract obtained by the current production process still need further improvement. Currently, the extraction of active ingredients from earthworms mainly uses methods such as salting out, ultrafiltration, and gel column chromatography. However, these methods are not suitable for industrial production and scale-up. For example, during crude extraction, buffer salt solutions are often used during homogenization to improve the dissolution of active ingredients, but desalting steps are required later. Alternatively, macroporous resin adsorption purification is used for extract purification. Although this method can significantly improve the purity of lumbrokinase and thus increase enzyme activity, the resin adsorption capacity is difficult to meet the needs of large-scale industrial production, and the cost is high, making it difficult to apply to the large-scale production of test samples.

[0005] Therefore, it is urgent to improve the extraction process of earthworms to further increase the extraction rate, purity, and enzyme activity of effective components such as lumbrokinase in earthworm extracts, thereby further enhancing the clinical efficacy of the drugs. Summary of the Invention

[0006] To achieve the above objectives, the present invention adopts the following technical solution: On one hand, the present invention provides an earthworm extract, wherein the earthworm extract includes an aprotinin; Preferably, the aprotinin content is not less than 4.5 mg / g; Preferably, the earthworm extract also includes lumbrokinase.

[0007] Preferably, the earthworm extract also includes lumbrokinase, and preferably, the lumbrokinase content is not less than 10 mg / g.

[0008] Preferably, the aprotinin content is 4.5 mg / g-55 mg / g, and the lumbrokinase content is 10 mg / g-90 mg / g; The aprotinin content is any value or range between 4.5 mg / g and 55 mg / g, specifically selectable from: 4.5 mg / g, 5 mg / g, 6 mg / g, 7 mg / g, 8 mg / g, 9 mg / g, 10 mg / g, 11 mg / g, 12 mg / g, 13 mg / g, 14 mg / g, 15 mg / g, 16 mg / g, 17 mg / g, 18 mg / g, 19 mg / g, 20 mg / g, 21 mg / g, 22 mg / g, 23 mg / g, 24 mg / g, 25 mg / g, 26 mg / g, 27 mg / g, 28 mg / g. The values ​​are g, 29mg / g, 30mg / g, 31mg / g, 32mg / g, 33mg / g, 34mg / g, 35mg / g, 36mg / g, 37mg / g, 38mg / g, 39mg / g, 40mg / g, 41mg / g, 42mg / g, 43mg / g, 44mg / g, 45mg / g, 46mg / g, 47mg / g, 48mg / g, 49mg / g, 50mg / g, 51mg / g, 52mg / g, 53mg / g, 54mg / g, 55mg / g, or a range between any two of the above values; The lumbrokinase content is any value or range between 10 mg / g and 90 mg / g, specifically selectable from: 10 mg / g, 11 mg / g, 12 mg / g, 13 mg / g, 14 mg / g, 15 mg / g, 16 mg / g, 17 mg / g, 18 mg / g, 19 mg / g, 20 mg / g, 21 mg / g, 22 mg / g, 23 mg / g, 24 mg / g, 25 mg / g, 26 mg / g, 27 mg / g, 28 mg / g, 29 mg / g, 30 mg / g, 31 mg / g, 32 mg / g, 33 mg / g, 34 mg / g, 35 mg / g, 36 mg / g, 37 mg / g, 38 mg / g, 39 mg / g, 40 mg / g, 41 mg / g, 42 mg / g, 43 mg / g, 44 mg / g, 45 mg / g, 46 ... mg / g, 47 mg / g, 48 mg / g, 49 mg / g, 50 mg / g, 51 mg / g, 52 mg / g, 53 mg / g, 54 mg / g, 55 mg / g, 56 mg / g, 57 mg / g, 58mg / g, 59 mg / g, 60 mg / g, 61 mg / g, 62 mg / g, 63 mg / g, 64 mg / g, 65 mg / g,66 mg / g,67 mg / g,68 mg / g,69 mg / g,70 mg / g,71 mg / g,72 mg / g,73 mg / g,74 mg / g,75 mg / g,76 mg / g,77 mg / g,78 mg / g,79 mg / g,80 mg / g,81 mg / g,82 mg / g,83 mg / g,84 mg / g, 85 mg / g, 86 mg / g, 87 mg / g, 88 mg / g, 89 mg / g or 90 mg / g, or a range between any two of the above values; Preferably, the content of the aprotinin is 15 mg / g-55 mg / g, and the content of the lumbrokinase is 20 mg / g-90 mg / g; More preferably, the content of the aprotinin is 30-55 mg / g, and the content of the lumbrokinase is 60-90 mg / g.

[0009] On the other hand, the present invention also provides an earthworm extract, wherein the earthworm extract comprises component A with a molecular weight <10kDa and component B with a molecular weight of 10-66kDa; Preferably, the distribution ratio of component A and component B in the earthworm extract is 40-90%: 10-60%; The distribution percentage of component A is any value or range between 40% and 90%, and can be 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or a range between any two of the above values; The distribution percentage of component B is any value or range between 10% and 60%, and can be 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or a range between any two of the above values.

[0010] More preferably, the distribution ratio of component A to component B in the earthworm extract is 40-65%:35-60%; More preferably, the ratio of component A to component B in the earthworm extract is 45-55%:45-55%; Furthermore, components A and B of the earthworm extract were determined by high-performance liquid chromatography using the peak area normalization method. Preferably, the high-performance liquid chromatography method is size exclusion chromatography, and more preferably gel chromatography. Furthermore, the determination methods for components A and B of the earthworm extract are as follows: A mixed reference solution is injected for analysis. Linear regression is performed using the retention time of each chromatographic peak in the mixed reference solution as the abscissa and the logarithm of the molecular weight as the ordinate to obtain the regression equation. A test sample solution is then injected for analysis. The retention times of each chromatographic peak are substituted into the regression equation to calculate the molecular weight distribution of the test sample. Based on the molecular weight distribution, the molecular weight ranges corresponding to components A and B are determined, and the proportion of substances in different molecular weight ranges is calculated using the peak area normalization method.

[0011] Furthermore, the determination methods for components A and B of the earthworm extract are as follows: Inject the above mixed reference solution for analysis. Using the retention time of each chromatographic peak in the mixed reference solution as the abscissa and the logarithm of the molecular weight as the ordinate, perform linear regression to obtain the regression equation Y = -0.0743X + 7.5047, r = 1. Inject the test sample solution for analysis, record the retention time of each chromatographic peak, substitute it into the regression equation, and calculate the molecular weight distribution of the test sample. Define the component with a molecular weight less than 10 kDa as component A, and define the component with a molecular weight in the range of 10-66 kDa as component B. Calculate the proportions of components A and B using the peak area normalization method.

[0012] Furthermore, the mixed control standards are bovine serum albumin, cytochrome C, and aprotinin; More preferably, the chromatographic conditions for determining components A and B of the earthworm extract are as follows: (1) Chromatographic conditions: Column: SHIMSEN Ankylo SEC-150 (5µm, 7.8×300mm); Mobile phase: Sodium chloride phosphate buffer; Flow rate: 0.3-0.8 mL / min; Column temperature: 20-30℃; Detection wavelength: 210-290nm; (2) Preparation of reference solution and test solution Preparation of the reference solution: Take appropriate amounts of bovine serum albumin reference standard, cytochrome C reference standard, and aprotinin reference standard, accurately weigh them, dissolve and dilute them with water to prepare a mixed solution containing approximately 10-1000 μg of each in 1 mL, as the mixed reference solution.

[0013] Take the above mixed reference solution and determine it according to the chromatographic conditions of (1). With the retention time of each chromatographic peak of the mixed reference solution as the abscissa and the logarithm of the molecular weight as the ordinate, perform linear regression to obtain the regression equation Y=-0.0743x+7.5047, r=1.

[0014] Preparation of the test solution: Take an appropriate amount of the aforementioned earthworm extract test sample, accurately weigh it, and prepare a test sample solution of 0.05-0.1 mg / mL. Perform liquid chromatography determination according to the chromatographic conditions in (1). Record the retention time of each chromatographic peak, substitute it into the regression equation, calculate the molecular weight distribution of the test sample, and calculate the proportion of substances in different molecular weight ranges using the normalization method.

[0015] On the other hand, the present invention also provides a method for preparing the above-mentioned earthworm extract, comprising the following steps: Weigh the earthworm and crush it into coarse powder. Add water and stir to form a homogenate. Combine the homogenates and add ethanol to make the alcohol content 5-25%. Let it stand for one alcohol precipitation and centrifuge. Take the supernatant, add ethanol to make the alcohol content 40-65%, let it stand for a second alcohol precipitation and centrifuge. Take the precipitate, wash it with propanol, centrifuge, and dry the precipitate at 40℃-70℃ to obtain the earthworm extract.

[0016] The amount of water added is 3-6 times the original amount; preferably, the amount of water added is 3.5-5.5 times the original amount; and even more preferably, the amount of water added is 4-5 times the original amount.

[0017] The amount of water added can be any value or range between 3 and 6 times the original amount, specifically selected from: 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, or any two of them.

[0018] The mixing and homogenization is performed 4-6 times, each time for 10-20 minutes, preferably 5 times, each time for 15 minutes.

[0019] The alcohol content of the primary alcohol precipitation is any value or range between 5% and 20%, specifically selected from: 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any two of these ranges.

[0020] Preferably, the alcohol content of the primary alcohol precipitation is 5-20%; more preferably, the alcohol content of the primary alcohol precipitation is 5-15%.

[0021] The temperature of the primary alcohol precipitation is 2-20℃; preferably, the temperature of the primary alcohol precipitation is 2-15℃; and even more preferably, the alcohol content of the primary alcohol precipitation is 2-10℃.

[0022] The temperature for the first alcohol precipitation is any value or range between 2 and 20°C, specifically selected from: 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, or any two of these ranges.

[0023] The time for the first alcohol precipitation is 6-24 hours; preferably, the time for the first alcohol precipitation is 8-18 hours; and even more preferably, the alcohol content of the first alcohol precipitation is 8-15 hours.

[0024] The time for the first alcohol precipitation is any value or range between 6 and 24 hours, specifically selected from: 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h or any two of them.

[0025] The alcohol content of the secondary alcohol precipitation is any value or range between 40% and 65%, specifically selected from: 40%, 45%, 50%, 55%, 60%, 65% or any two of them.

[0026] Preferably, the alcohol content of the secondary alcohol precipitation is 40-60%; more preferably, the alcohol content of the secondary alcohol precipitation is 45-55%.

[0027] The temperature of the secondary alcohol precipitation is 2-20℃; preferably, the temperature of the secondary alcohol precipitation is 2-15℃; and even more preferably, the alcohol content of the secondary alcohol precipitation is 2-10℃.

[0028] The temperature for the secondary alcohol precipitation is any value or range between 2 and 20°C, specifically selected from: 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, or any two of these ranges.

[0029] The secondary alcohol precipitation time is 6-24 hours; preferably, the secondary alcohol precipitation time is 8-18 hours; and even more preferably, the alcohol content of the secondary alcohol precipitation is 8-15 hours.

[0030] The time for the secondary alcohol precipitation is any value or range between 6 and 24 hours, specifically selected from: 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h or any two of these ranges.

[0031] The drying temperature is any value or range between 40-70℃, specifically selected from: 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃ or any two of them.

[0032] Preferably, the drying temperature is 55-65°C; Preferably, the drying process includes, but is not limited to, vacuum drying, freeze drying, and spray drying. Preferably, the drying is vacuum drying; the pressure of the vacuum drying is 0.06-0.10 MPa; preferably 0.08 MPa.

[0033] Furthermore, the present invention also provides an earthworm extract, which is prepared by the following method: Weigh the earthworm and crush it into coarse powder. Add water and stir to form a homogenate. Combine the homogenates and add ethanol to make the alcohol content 5-25%. Let it stand for one alcohol precipitation. Take the supernatant and add ethanol to make the alcohol content 40-65%. Let it stand for a second alcohol precipitation. Dry the precipitate and wash it with propanol. Centrifuge the precipitate and dry it at 40℃-70℃ to obtain the earthworm extract.

[0034] The amount of water added is 3-6 times the original amount; preferably, the amount of water added is 3.5-5.5 times the original amount; and even more preferably, the amount of water added is 4-5 times the original amount.

[0035] The amount of water added can be any value or range between 3 and 6 times the original amount, specifically selected from: 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, or any two of them.

[0036] The mixing and homogenization is performed 4-6 times, each time for 10-20 minutes, preferably 5 times, each time for 15 minutes.

[0037] The alcohol content of the primary alcohol precipitation is any value or range between 5% and 20%, specifically selected from: 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any two of these ranges.

[0038] Preferably, the alcohol content of the primary alcohol precipitation is 5-20%; more preferably, the alcohol content of the primary alcohol precipitation is 5-15%.

[0039] The temperature of the primary alcohol precipitation is 2-20℃; preferably, the temperature of the primary alcohol precipitation is 2-15℃; and even more preferably, the alcohol content of the primary alcohol precipitation is 2-10℃.

[0040] The temperature for the first alcohol precipitation is any value or range between 2 and 20°C, specifically selected from: 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, or any two of these ranges.

[0041] The time for the first alcohol precipitation is 6-24 hours; preferably, the time for the first alcohol precipitation is 8-18 hours; and even more preferably, the alcohol content of the first alcohol precipitation is 10-15 hours.

[0042] The time for the first alcohol precipitation is any value or range between 6 and 24 hours, specifically selected from: 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h or any two of them.

[0043] The alcohol content of the secondary alcohol precipitation is any value or range between 40% and 65%, specifically selected from: 40%, 45%, 50%, 55%, 60%, 65% or any two of them.

[0044] Preferably, the alcohol content of the secondary alcohol precipitation is 40-60%; more preferably, the alcohol content of the secondary alcohol precipitation is 45-55%.

[0045] The temperature of the secondary alcohol precipitation is 2-20℃; preferably, the temperature of the secondary alcohol precipitation is 2-15℃; and even more preferably, the alcohol content of the secondary alcohol precipitation is 2-10℃.

[0046] The temperature for the secondary alcohol precipitation is any value or range between 2 and 20°C, specifically selected from: 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, or any two of these ranges.

[0047] The secondary alcohol precipitation time is 6-24 hours; preferably, the secondary alcohol precipitation time is 8-18 hours; and even more preferably, the alcohol content of the secondary alcohol precipitation is 10-15 hours.

[0048] The time for the secondary alcohol precipitation is any value or range between 6 and 24 hours, specifically selected from: 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h or any two of these ranges.

[0049] The drying temperature is any value or range between 40-70℃, specifically selected from: 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃ or any two of them.

[0050] Preferably, the drying temperature of the precipitate is 55-65°C; Preferably, the drying process includes, but is not limited to, vacuum drying, freeze drying, and spray drying. Preferably, the drying is vacuum drying; the pressure of the vacuum drying is 0.06-0.10 MPa; preferably 0.08 MPa.

[0051] In another aspect, the present invention also provides a traditional Chinese medicine extraction composition, wherein the extraction composition comprises earthworm extract obtained from earthworms through the above-described preparation.

[0052] The extraction composition also includes: processed Polygonum multiflorum, lotus leaf, Cistanche deserticola, and Echinops latifolius.

[0053] As a preferred embodiment, the extraction composition comprises, by weight, the following components: 20-50 parts of processed Polygonum multiflorum, 10-30 parts of lotus leaf, 10-30 parts of Cistanche deserticola, 8-25 parts of earthworm, and 2-20 parts of Echinops latifolius. Preferably, the extraction composition comprises, by weight parts: 20-40 parts of processed Polygonum multiflorum, 20-30 parts of lotus leaf, 10-25 parts of Cistanche deserticola, 10-25 parts of earthworm, and 5-20 parts of Echinops latifolius. More preferably, the extraction composition comprises, by weight parts: 25-35 parts of processed Polygonum multiflorum, 20-25 parts of lotus leaf, 15-20 parts of Cistanche deserticola, 15-20 parts of earthworm, and 8-15 parts of Echinops latifolius. Most preferably, in the extraction composition, the raw materials are 35 parts by weight of processed Polygonum multiflorum, 24 parts of lotus leaf, 17 parts of Cistanche deserticola, 17 parts of earthworm, and 10 parts of Echinops latifolius.

[0054] Further, the preparation method of the extraction composition includes: weighing the following raw materials according to the following weight parts for preparation: 20-50 parts of prepared Polygonum multiflorum, 10-30 parts of lotus leaf, 10-30 parts of Cistanche deserticola, 2-20 parts of Echinops latifolius, and 8-25 parts of earthworm. The prepared Polygonum multiflorum, lotus leaf, Cistanche deserticola, and Echinops latifolius are pulverized, and water or ethanol is added for extraction or extraction separately. The mixture is then combined with the earthworm extract or the earthworm extract prepared by the aforementioned preparation method to obtain the final product.

[0055] More preferably, the method for preparing the extraction composition includes the following steps: (1) Weigh the prepared Polygonum multiflorum, crush it, extract it by heating and reflux with ethanol, combine the filtrates, recover the ethanol under reduced pressure, and concentrate it into a paste; (2) Boil lotus leaves, Cistanche deserticola and Echinops latifolius in water, combine the decoctions, concentrate the decoction to a relative density of 1.0-1.3 (50-80℃), cool and add ethanol, let stand, filter, recover the ethanol from the filtrate, and concentrate under reduced pressure to a clear extract with a relative density of 1.0-1.5 (50-80℃). (3) Combine the clear pastes obtained in steps (1) and (2), and vacuum dry them to make a dry paste; (4) Mix the dry paste obtained in step (3) with the earthworm extract prepared by the above method to obtain the extraction composition.

[0056] The particle size of the pulverized material in step (1) above is 8-50 mm; preferably 30 mm.

[0057] The concentration of ethanol mentioned in step (1) above is 60-90%; preferably 75%.

[0058] The extraction process described in step (1) above is performed 1-3 times; The extraction in step (1) above is performed twice. The solvent added for the first extraction is 6-15 times the amount of solvent and the extraction time is 1-3 hours. The solvent added for the second extraction is 6-15 times the amount of solvent and the extraction time is 1-3 hours.

[0059] In step (1) above, the concentration is carried out at 50-80℃ until the relative density is 1.0-1.5; The number of times to decoct in step (2) above is 1-3; The decoction process described in step (2) above is performed twice. The amount of solvent added for the first decoction is 8-15 times the amount of solvent, and the time is 2-3 hours. The amount of solvent added for the second decoction is 6-12 times the amount of solvent, and the time is 2-3 hours.

[0060] In step (2) above, the ethanol concentration is increased to 40%-70%, preferably 50%-60%, and the standing time is 8-24 hours. In another aspect, the present invention also provides a pharmaceutical preparation comprising the above-mentioned traditional Chinese medicine extract composition and pharmaceutically acceptable excipients.

[0061] Preferably, the pharmaceutical preparation includes granules, tablets, powders, capsules, oral liquids, injections, sprays, aerosols, powder inhalers, eye drops, nasal drops, suppositories, pills, microspheres, or tinctures.

[0062] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, lubricants, disintegrants, flow aids, and wetting agents; More preferably, the pharmaceutical preparation is a capsule, and the excipients acceptable in the capsule include, but are not limited to, fillers, binders, lubricants, disintegrants, flow aids, and wetting agents; Furthermore, the present invention also provides a method for preparing the above-mentioned capsule formulation, which involves pulverizing and sieving the above-mentioned traditional Chinese medicine extract composition, adding excipients, and filling it into capsules.

[0063] In another aspect, the present invention provides a method for quantitative detection of aprotinin in capsule formulations, wherein the detection method is determined by external standard method of high performance liquid chromatography.

[0064] Furthermore, the detection method includes the following steps: preparing a reference standard and a test solution, injecting them into a high-performance liquid chromatograph, and measuring them to obtain the result.

[0065] Furthermore, the quantitative detection method for aprotinin in the capsule formulation includes the following steps: Preparation of reference and test solutions: Accurately weigh appropriate amounts of aprotinin and capsule contents, add acetonitrile and a formic acid solution to prepare a solution. Inject the solution into a high-performance liquid chromatograph and determine the result.

[0066] The quantitative detection results of the capsule formulation showed that the aprotinin content in the capsule formulation was not less than 0.4 mg / g; Preferably, the aprotinin content in the capsule formulation is not less than 0.5 mg / g; More preferably, the aprotinin content in the capsule formulation is 0.58-1.54 mg / g.

[0067] Furthermore, the present invention also provides the application of the above-mentioned traditional Chinese medicine extract composition or pharmaceutical preparation in the preparation of a drug for treating schizophrenia; wherein the schizophrenia includes, but is not limited to, negative symptoms, positive symptoms, and cognitive impairment; Furthermore, the negative symptoms include, but are not limited to, emotional apathy, poverty of thought, diminished will, social withdrawal, and anhedonia; the positive symptoms include, but are not limited to, hallucinations, delusions, and disordered thinking; and the cognitive impairments include, but are not limited to, attention deficit, impaired working memory, slowed information processing, and learning and memory disorders.

[0068] Furthermore, the present invention also provides the application of the above-mentioned traditional Chinese medicine extract composition or pharmaceutical preparation in the preparation of pharmaceutical preparations for treating cognitive impairment; preferably, the cognitive impairment is selected from one or more of Alzheimer's disease, vascular dementia, mild cognitive impairment, frontotemporal dementia, Lewy body dementia, Parkinson's disease dementia, and other types of dementia. Furthermore, the present invention also provides the application of the above-mentioned traditional Chinese medicine extract composition or pharmaceutical preparation in the preparation of a drug for treating cognitive impairment in schizophrenia, wherein the cognitive impairment includes, but is not limited to, attention deficit, impaired working memory, slowed information processing speed, learning and memory impairment, etc.

[0069] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides an earthworm extract comprising component A with a molecular weight of about <10kDa and component B with a molecular weight of about 10-66kDa, wherein the earthworm extract further comprises aprotinin and / or lumbrokinase, and the earthworm extract has superior enzyme activity and extremely strong anticoagulant efficacy. This invention optimizes the earthworm preparation process, especially by optimizing the process steps and parameters of alcohol precipitation and drying, thereby improving the extraction rate of earthworm protein and resulting in an extract with higher enzyme activity and anticoagulant effect. Using specific qualitative and quantitative detection methods, earthworm extract was found to contain lumbrokinase and aprotinin. This invention is the first to detect aprotinin in earthworm and establishes other evaluation indicators for earthworm extract besides enzyme activity. This invention also provides the application of traditional Chinese medicine extract compositions or pharmaceutical preparations in the preparation of drugs for treating cognitive impairment, schizophrenia, and cognitive impairment in schizophrenia. The traditional Chinese medicine extract compositions or pharmaceutical preparations containing optimized earthworm extracts have significantly better therapeutic effects on cognitive impairment than those before process optimization, and provide clinical data support for drugs for schizophrenia and cognitive impairment in schizophrenia, providing more options for clinical patients. This invention also provides a qualitative and quantitative detection method for aprotinin in capsule formulations, which provides a foundation for quality control of capsule formulations and improves product quality evaluation standards. Attached Figure Description

[0070] Figure 1 From top to bottom, the images show a comparison of the HPLC chromatographic peak retention times of the aprotinin reference standard and the test sample. Figure 2 The images show the UV absorption spectra of the chromatographic peaks at corresponding retention times of the aprotinin reference standard and the test sample under different chromatographic conditions. From top to bottom, the images represent chromatographic conditions 1, 2, and 3. The left side shows the UV absorption spectrum of the aprotinin standard, and the right side shows the UV absorption spectrum of the corresponding absorption peak of the test sample. Figure 3 The colorimetric identification diagram for aprotinin is as follows: 1. Trypsin + reagent; 2. Aprotinin + trypsin + reagent; 3-5 are, respectively, the test samples from Examples 1-3 + trypsin + reagent; 6. Blank solvent (water) + trypsin + reagent. The reagent refers to p-toluenesulfonyl-L-arginine methyl ester hydrochloride reagent. Figure 4 The brain regions where the FA value interaction was significant in Example 13 of Example 4; Figure 5 The change in FA value in the differential brain regions in Example 13 of Example 4; Detailed Implementation The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0071] The experimental materials and instruments are shown in Tables 1 and 2.

[0072] Table 1 Experimental Instruments

[0073] Table 2 Experimental Reagents

[0074] Example 1: An earthworm extract and its preparation method The earthworm extract was prepared by the following method: 25g of earthworm was weighed and crushed into coarse powder, 4 times the amount of water was added and stirred into a homogenate 5 times, 15min each time, the homogenates were combined, ethanol was added to make the alcohol content 10%, and the mixture was allowed to stand at 10℃ for a first alcohol precipitation for 15h, centrifuged, and the supernatant was obtained; the supernatant was taken and ethanol was added to make the alcohol content 50%, centrifuged, and allowed to stand at 8℃ for a second alcohol precipitation for 12h, the precipitate was washed with acetone, centrifuged, and the precipitate was dried under vacuum at 60℃ and 0.08MPa to obtain the earthworm extract.

[0075] Example 2: An earthworm extract and its preparation method The earthworm extract was prepared by the following method: 25g of earthworm was weighed and crushed into coarse powder, 3.5 times the amount of water was added and stirred into a homogenate 6 times, 15min each time. The homogenates were combined, and ethanol was added to make the alcohol content 12%. The mixture was allowed to stand at 8°C for a first alcohol precipitation for 8h, centrifuged, and the supernatant was obtained. The supernatant was taken and ethanol was added to make the alcohol content 45%, centrifuged, and allowed to stand at 10°C for a second alcohol precipitation for 10h. The precipitate was washed with acetone, centrifuged, and the precipitate was spray-dried at 65°C to obtain the earthworm extract.

[0076] Example 3: An earthworm extract and its preparation method The earthworm extract was prepared by the following method: 25g of earthworm was weighed and crushed into coarse powder, 4 times the amount of water was added and stirred into a homogenate 4 times, 15min each time, the homogenates were combined, ethanol was added to make the alcohol content 15%, and the mixture was allowed to stand at 2℃ for a first alcohol precipitation for 10h, centrifuged, and the supernatant was obtained; the supernatant was taken and ethanol was added to make the alcohol content 55%, centrifuged, and allowed to stand at 5℃ for a second alcohol precipitation for 8h, the precipitate was washed with acetone, centrifuged, and the precipitate was dried under vacuum at 60℃ and 0.06MPa to obtain the earthworm extract.

[0077] Example 4: An earthworm extract and its preparation method The earthworm extract was prepared by the following method: 25g of earthworm was weighed and crushed into coarse powder, and 3 times the amount of water was added and stirred into a homogenate slurry 6 times, 15min each time. The homogenates were combined, and ethanol was added to make the alcohol content 5%. The mixture was allowed to stand at 5℃ for a first alcohol precipitation for 12h, and centrifuged to obtain the supernatant. The supernatant was then added to make the alcohol content 55%, centrifuged, and allowed to stand at 2℃ for a second alcohol precipitation for 15h. The precipitate was washed with acetone, centrifuged, and vacuum dried at 55℃ and 0.1MPa to obtain the earthworm extract.

[0078] Example 5: An earthworm extract and its preparation method The earthworm extract was prepared by the following method: 25g of earthworm was weighed and crushed into coarse powder, 4.5 times the amount of water was added and stirred into a homogenate 5 times, 15min each time. The homogenates were combined, and ethanol was added to make the alcohol content 25%. The mixture was allowed to stand at 12℃ for a first alcohol precipitation for 6h, centrifuged, and the supernatant was obtained. The supernatant was taken and ethanol was added to make the alcohol content 40%, centrifuged, and allowed to stand at 20℃ for a second alcohol precipitation for 16h. The precipitate was washed with acetone, centrifuged, and the precipitate was vacuum dried at 45℃ and 0.08MPa to obtain the earthworm extract.

[0079] Example 6: An earthworm extract and its preparation method The earthworm extract was prepared by the following method: 25g of earthworm was weighed and crushed into coarse powder, 5.5 times the amount of water was added and stirred into a homogenate 4 times, 15min each time. The homogenates were combined, and ethanol was added to make the alcohol content 22%. The mixture was allowed to stand at 20℃ for a first alcohol precipitation for 24h, centrifuged, and the supernatant was obtained. The supernatant was taken and ethanol was added to make the alcohol content 65%, centrifuged, and allowed to stand at 15℃ for a second alcohol precipitation for 20h. The precipitate was washed with acetone, centrifuged, and the precipitate was vacuum dried at 60℃ and 0.1MPa to obtain the earthworm extract.

[0080] Example 7: An earthworm extract and its preparation method The earthworm extract was prepared by the following method: 25g of earthworm was weighed and crushed into coarse powder, 5.5 times the amount of water was added and stirred into a homogenate 6 times, 15min each time. The homogenates were combined, and ethanol was added to make the alcohol content 20%. The mixture was allowed to stand at 10℃ for a first alcohol precipitation for 18h, centrifuged, and the supernatant was obtained. The supernatant was taken and ethanol was added to make the alcohol content 60%, centrifuged, and allowed to stand at 5℃ for a second alcohol precipitation for 12h. The precipitate was washed with acetone, centrifuged, and the precipitate was vacuum dried at 40℃ and 0.08MPa to obtain the earthworm extract.

[0081] Example 8: An earthworm extract and its preparation method The earthworm extract was prepared by the following method: 25g of earthworm was weighed and crushed into coarse powder, 5 times the amount of water was added and stirred into a homogenate 5 times, 15min each time, the homogenates were combined, ethanol was added to make the alcohol content 5%, and the mixture was allowed to stand at 2℃ for a first alcohol precipitation for 10h, centrifuged, and the supernatant was obtained; the supernatant was taken and ethanol was added to make the alcohol content 65%, centrifuged, and allowed to stand at 5℃ for a second alcohol precipitation for 6h, the precipitate was washed with acetone, centrifuged, and the precipitate was vacuum dried at 55℃ and 0.08MPa to obtain the earthworm extract.

[0082] Example 9: An earthworm extract and its preparation method The earthworm extract was prepared by the following method: 25g of earthworm was weighed and crushed into coarse powder, 6 times the amount of water was added and stirred into a homogenate 4 times, 15min each time, the homogenates were combined, ethanol was added to make the alcohol content 16%, and the mixture was allowed to stand at 15℃ for a first alcohol precipitation for 16h, centrifuged, and the supernatant was obtained; the supernatant was taken and ethanol was added to make the alcohol content 65%, centrifuged, and allowed to stand at 10℃ for a second alcohol precipitation for 18h, the precipitate was washed with acetone, centrifuged, and the precipitate was vacuum dried at 70℃ and 0.08MPa to obtain the earthworm extract.

[0083] Example 10: An earthworm extract and its preparation method The earthworm extract was prepared by the following method: 25g of earthworm was weighed and crushed into coarse powder, 4.5 times the amount of water was added and stirred into a homogenate 6 times, 15min each time. The homogenates were combined, and ethanol was added to make the alcohol content 18%. The mixture was allowed to stand at 20℃ for a first alcohol precipitation for 20h, centrifuged, and the supernatant was obtained. Ethanol was added to the supernatant to make the alcohol content 60%, centrifuged, and allowed to stand at 18℃ for a second alcohol precipitation for 16h. The precipitate was washed with acetone, centrifuged, and the precipitate was vacuum dried at 65℃ and 0.08MPa to obtain the earthworm extract.

[0084] Example 11: An earthworm extract and its preparation method The earthworm extract was prepared by the following method: 25g of earthworm was weighed and crushed into coarse powder, 4 times the amount of water was added and stirred into a homogenate 6 times, 15min each time, the homogenates were combined, ethanol was added to make the alcohol content 10%, and the mixture was allowed to stand at 15℃ for a first alcohol precipitation for 20h, centrifuged, and the supernatant was obtained; the supernatant was taken and ethanol was added to make the alcohol content 55%, centrifuged, and allowed to stand at 10℃ for a second alcohol precipitation for 24h, the precipitate was washed with acetone, centrifuged, and the precipitate was vacuum dried at 60℃ and 0.08MPa to obtain the earthworm extract.

[0085] Comparative Example 1 The difference from Example 1 is that the temperature of the first alcohol precipitation is 0°C and the time is 2 hours, and the temperature of the second alcohol precipitation is 0°C and the time is 30 hours. Everything else is the same as in Example 1.

[0086] Comparative Example 2 The difference from Example 1 is that the alcohol content of the first alcohol precipitation is 2% and the time is 5 hours, while the alcohol content of the second alcohol precipitation is 35%, the temperature is 20°C, and the time is 30 hours. The rest is the same as Example 1.

[0087] Comparative Example 3 The difference from Example 1 is that the temperature of the second alcohol precipitation is 10°C and the drying temperature is 75°C, while the rest is the same as in Example 1.

[0088] Comparative Example 4 The difference from Example 1 is that the alcohol content of the first alcohol precipitation is 15%, and the alcohol content of the second alcohol precipitation is 35%, while the rest is the same as in Example 1.

[0089] Comparative Example 5 The difference from Example 1 is that the temperature of the first alcohol precipitation is 25°C and the temperature of the second alcohol precipitation is 0°C, while the rest is the same as Example 1.

[0090] Comparative Example 6 The difference from Example 1 is that the first alcohol precipitation time is 5 hours, the second alcohol precipitation temperature is 10°C and the time is 30 hours, and the rest is the same as Example 1.

[0091] Comparative Example 7 The difference from Example 1 is that the alcohol content of the first alcohol precipitation is 30%, and the alcohol content of the second alcohol precipitation is 55%, while the rest is the same as in Example 1.

[0092] Comparative Example 8 The difference from Example 1 is that the alcohol content of the first alcohol precipitation is 20% and the temperature is 20°C, and the alcohol content of the second alcohol precipitation is 70% and the temperature is 25°C. The rest is the same as Example 1.

[0093] Example 12 Detection method for earthworm extract The detection methods include molecular weight distribution detection of earthworm extract, qualitative detection of aprotinin, and quantitative detection of earthworm extract indicator components lumbrokinase and aprotinin.

[0094] Method for determining the molecular weight distribution of earthworm extract Experimental methods (1) Chromatographic conditions: Column: SHIMSEN Ankylo SEC-150 (5µm, 7.8×300mm); Mobile phase: 50 mM phosphate buffer containing 300 mM sodium chloride; Flow rate: 0.2 mL / min; Column temperature: 30℃; Detection wavelength: 214nm; Injection volume: 5µL; (2) Preparation of reference standard and test solution Preparation of the reference solution: Take appropriate amounts of bovine serum albumin reference standard, cytochrome C reference standard, and aprotinin reference standard, accurately weigh them, dissolve and dilute them with water to prepare a mixed solution containing approximately 100 μg of each in 1 mL, as the mixed reference solution. Take the above mixed reference solution and determine it according to the chromatographic conditions in (1). With the retention time of each chromatographic peak of the mixed reference solution as the abscissa and the logarithm of the molecular weight as the ordinate, perform linear regression to obtain the regression equation Y = -0.0743x + 7.5047, r = 1.

[0095] Preparation of the test solution: Take appropriate amounts of earthworm extract samples prepared in Examples 1-11 and Comparative Examples 1-8, accurately weigh them, and prepare a 1 mg / mL solution. Perform liquid chromatography analysis according to the chromatographic conditions in (1). Each sample is tested in triplicate. Record the retention time of each chromatographic peak, substitute it into the regression equation, calculate the molecular weight distribution of the sample, and calculate the proportion of substances in different molecular weight ranges using the normalization method.

[0096] Test results Table 1. Molecular weight distribution of earthworm extracts prepared in Examples 1-11 and Comparative Examples 1-8 (n=3)

[0097] Testing revealed that the earthworm extracts obtained by the preparation process in Examples 1-11 of this invention have molecular weight distributions of 10-66 kDa and <10 kDa, indicating that the process can effectively remove high molecular weight substances with a molecular weight greater than 66 kDa.

[0098] Qualitative Detection Method of Aprotinin, an Indicator Component of Earthworm Extract (1) Retention time comparison: Using the retention time of the aprotinin reference standard as a reference, the ratio of the retention time of the corresponding chromatographic peak of the earthworm extract prepared in Example 1 to the retention time of the aprotinin reference standard was calculated under different chromatographic conditions, and the RSD of the retention time ratio was calculated.

[0099] Chromatographic conditions 1: Column: SHIMSEN Ankylo SEC-150 (5µm, 7.8×300mm); Mobile phase: 50mM phosphate buffer containing 300mM sodium chloride; Flow rate: 0.2mL / min; Chromatographic conditions 2: Column: SHIMSEN AnkyloSEC-150 (5µm, 7.8×300mm); Mobile phase: 50mM phosphate buffer containing 300mM sodium chloride; Flow rate: 0.5mL / min; Chromatographic conditions 3: Column: SHIMSEN Ankylo SEC-150 (5µm, 7.8×300mm); Mobile phase: 2% acetonitrile: 98% aqueous solution containing 0.15% formic acid; Flow rate: 0.6mL / min. Detection results are shown in Table 2 below.

[0100] Table 2. HPLC peak retention times of aprotinin reference standard and test sample under different chromatographic conditions.

[0101] According to the test results in Table 2, under different chromatographic conditions, the RSD of the ratio of the retention time of the test sample aprotinin chromatographic peak to the retention time of the aprotinin reference standard was 1.77%, indicating that the method of using relative retention time to qualitatively identify chromatographic peaks is feasible.

[0102] (2) Comparison of ultraviolet absorption spectra: The UV absorption spectra of the aprotinin reference standard under different chromatographic conditions were compared with the UV absorption spectra of the earthworm extract test sample prepared in Example 1 at the corresponding retention time peaks.

[0103] Chromatographic conditions 1: Column: SHIMSEN Ankylo SEC-150 (5µm, 7.8×300mm); Mobile phase: 50mM phosphate buffer containing 300mM sodium chloride; Flow rate: 0.2mL / min; Chromatographic conditions 2: Column: SHIMSEN AnkyloSEC-150 (5µm, 7.8×300mm); Mobile phase: 50mM phosphate buffer containing 300mM sodium chloride; Flow rate: 0.5mL / min; Chromatographic conditions 3: Column: SHIMSEN Ankylo SEC-150 (5µm, 7.8×300mm); Mobile phase: 2% acetonitrile: 98% aqueous solution containing 0.15% formic acid; Flow rate: 0.6mL / min.

[0104] according to Figure 1It can be seen that under chromatographic condition 3, the UV absorption spectra of the corresponding retention time positions of the aprotinin reference standard and the test sample both exhibit a characteristic absorption peak at 278 nm, and the peak shapes are similar. Under chromatographic conditions 1 and 2, the UV absorption spectra of the corresponding retention time positions of the aprotinin reference standard and the test sample also show the same characteristic absorption peak and similar absorption peak shapes at the corresponding retention times.

[0105] (3) Aprotinin colorimetric reaction: According to the identification item of aprotinin quality standard in the Chinese Pharmacopoeia, the earthworm extract samples prepared in Examples 1-3 were subjected to colorimetric identification reactions.

[0106] Preparation of test solution: Accurately weigh the earthworm extract test sample, add water to prepare a 1 mg / mL test solution, and filter through a 0.22 μm microporous membrane to obtain the solution; Assay: Dissolve and dilute the earthworm extract and trypsin prepared in Examples 1-3 separately in water to prepare solutions containing 1 mg per mL. Place 10 μl of each solution on a spot plate, mix well, and add 0.2 mL of p-toluenesulfonyl-L-arginine methyl ester hydrochloride solution. Observe the color development after standing for several minutes. Use 10 μl of trypsin solution as a control on a spot plate, mix well, and add 0.2 mL of p-toluenesulfonyl-L-arginine methyl ester hydrochloride solution. Observe the color development after standing for several minutes. Separately, place 10 μl of blank water on a spot plate, add 10 μl of trypsin solution, mix well, add 0.2 mL of p-toluenesulfonyl-L-arginine methyl ester hydrochloride solution, and observe the color development after standing for several minutes.

[0107] according to Figure 2 It can be seen that at the same time point, test sample No. 1 (trypsin control) and test sample No. 6 (blank solvent (water) + trypsin control) showed a purple-red color, while test sample No. 2 (aprotinin control) did not show a purple-red color. The color development results of test samples No. 3-5 (Examples 1-3) were consistent with those of the aprotinin control, indicating that the test sample and the aprotinin control have the same trypsin inhibitory effect.

[0108] In summary, after multi-dimensional verification, the chromatographic peaks of the test sample are consistent with the retention time and ultraviolet spectrum of the aprotinin reference standard, and the colorimetric reaction meets the colorimetric reaction requirements of the identification item of the aprotinin quality standard in the Chinese Pharmacopoeia (2025 edition).

[0109] Quantitative detection of lumbrokinase and aprotinin, key components of earthworm extract Detection methods (1) Preparation of reference solutions: Accurately weigh lumbrokinase reference standard and aprotinin reference standard, add 2% acetonitrile and 98% aqueous solution containing 0.15% formic acid respectively to prepare solutions of 100 μg / mL.

[0110] (2) Preparation of test solution: Weigh approximately 10 mg of earthworm extract prepared in Examples 1-11 and Comparative Examples 1-8, place it in a 10 mL volumetric flask, add 2% acetonitrile and 98% aqueous solution containing 0.15% formic acid to the mark, weigh, sonicate for 10 min, cool, replenish the lost weight with 2% acetonitrile and 98% aqueous solution containing 0.15% formic acid, shake well, and filter through a 0.22 µm microporous membrane to obtain the test solution.

[0111] (3) Chromatographic conditions: SHIMSEN Ankylo SEC-150 (5µm, 7.8×300mm); mobile phase: 2% acetonitrile: 98% aqueous solution containing 0.15% formic acid; flow rate: 0.6mL / min; detection wavelength: 280nm; injection volume: 5µL.

[0112] The concentration of the target component in the test sample is calculated using the external standard method formula Cstandard / Astandard = Csample / Asample, and the content is calculated accordingly.

[0113] Test results Table 3. Contents of lumbrokinase and aprotinin in earthworm extracts prepared in Examples 1-11 and Comparative Examples 1-8 (n=3)

[0114] According to the test results in Table 3 above, the earthworm extract prepared in the embodiments of the present invention has a high content of lumbrokinase and aprotinin, indicating that the earthworm extract preparation method provided by the present invention can effectively classify lumbrokinase and aprotinin in earthworms and has high production efficiency.

[0115] Example 1: Evaluation of the in vitro thrombolytic activity of earthworm extract Enzyme activity was determined using the fibrin plate method.

[0116] Preparation of fibrinogen plates: Take 39 mL of fibrinogen solution and place it in a beaker. While stirring, add 39 mL of 55℃ agarose solution and 3.0 mL of thrombin solution. Mix well immediately and quickly pour into a 15 cm diameter plastic petri dish. Place horizontally for 1 hour and punch holes using a punch with an inner diameter of 3 mm. Preparation of standard solutions: Take lumbrokinase reference standard and add 0.9% sodium chloride solution to prepare solutions with concentrations of 500, 1000, 2000, 3000, and 5000 U / mL lumbrokinase units.

[0117] Preparation method of test solution: Accurately weigh earthworm extract, add 5 mL of 0.9% sodium chloride solution, sonicate for 10 minutes, centrifuge at 4500 rpm for 5 minutes, and take the supernatant. Repeat in triplicate.

[0118] Assay: Accurately measure 20 μL each of the series concentrations of lumbrokinase standard solution, test solution, and blank solution (0.9% sodium chloride solution), add them separately to the test sample wells of the same petri dish, duplicate the wells, cap, and incubate at 37℃ for 18 hours. After removal, measure the two perpendicular diameters of the melting zone with calipers. Plot the logarithm of the number of units of lumbrokinase standard solution on the x-axis and the logarithm of the product of the two perpendicular diameters on the y-axis, calculate the regression equation (requiring a correlation coefficient R≥0.98). Substitute the logarithm of the product of the two perpendicular diameters of the test sample into the regression equation, and calculate the lumbrokinase potency per 1 mg of test sample using the average value. The results are shown in Table 4.

[0119] Table 4. Evaluation results of the in vitro thrombolytic activity of earthworm extract (n=3)

[0120] As can be seen from the test results in Table 4 above, the earthworm extracts prepared in Examples 1-11 of the present invention have higher specific activity, that is, higher purity and better enzyme activity, and are significantly better than those in Comparative Examples 1-8, indicating that the earthworm extracts of the present invention are significantly more effective.

[0121] Example 2: Determination of thromboplastin time (APTT method) using earthworm extract. Plasma preparation: Quickly collect rabbit blood and place it in a container pre-filled with 109 mmol / L sodium citrate solution (sodium citrate solution to rabbit blood volume ratio 1:9). Gently shake while collecting to mix well, and centrifuge at 1500×g for 20 minutes at room temperature. Immediately aspirate the plasma and aliquot it into centrifuge tubes, storing at -20℃. Before use, thaw the plasma in a 37℃±0.5℃ water bath, filter through two layers of gauze or rapid filter paper, and store at 4-8℃ during use.

[0122] Preparation of test solution: Weigh three batches of earthworm extract prepared in the examples and comparative examples respectively, add 5.0 mL of 0.9% sodium chloride solution, sonicate for 10 min, centrifuge for 5 min and take the supernatant, and take the supernatant as the test solution.

[0123] Assay: The APTT method was used. Several sample cups from the coagulation analyzer were taken, and 50 μL of plasma, 50 μL of 0.5 IU / mL heparin sodium or the test solution, and 50 μL of APTT reagent were added to each tube sequentially, and mixed well. After preheating at 37℃±0.5℃ for 180 seconds, 50 μL of 0.025 mol CaCl2 reagent was added to each tube, and the clotting time (activated partial thromboplastin time, APTT) was immediately measured using the coagulation analyzer. The assay was performed in triplicate. A 0.9% sodium chloride solution was used as a blank control. A 0.9% sodium chloride solution was used as a blank control, and heparin sodium was used as a positive control. The assays were performed in triplicate. The results are shown in Table 5.

[0124] Table 5. Results of thromboplastin time assay for earthworm extract (n=3)

[0125] As can be seen from the test results in the table above, the earthworm extract prepared in this embodiment of the invention has a longer thromboplastin time.

[0126] Example 13: A capsule formulation and its preparation method The preparation method is as follows: (1) Weigh 35 parts of processed Polygonum multiflorum, crush it into coarse particles with a diameter of 30 mm, and extract it twice by heating and reflux with 75% ethanol. The amount of ethanol used in the first extraction is 8 times the amount of medicinal material and the extraction time is 2 hours. The amount of ethanol used in the second extraction is 6 times the amount of medicinal material and the extraction time is 2 hours. Combine the filtrates, recover the ethanol under reduced pressure, and concentrate it to a paste with a relative density of 1.3 at 60℃. (2) Add 25 parts of lotus leaf, 15 parts of Cistanche deserticola and 5 parts of Echinops latifolius to water and decoct twice. The first time, add 10 times the amount of water and decoct for 2 hours. The second time, add 8 times the amount of water and decoct for 2 hours. Combine the decoctions and concentrate them to a relative density of 1.3 (60℃). After cooling, add ethanol to make the alcohol content reach 40%. Let stand for 12 hours, filter, recover the ethanol from the filtrate, and concentrate under reduced pressure to a clear extract with a relative density of 1.30 (60℃). (3) Combine the clear pastes obtained in steps (1) and (2), and vacuum dry them to make a dry paste; (4) Earthworm extract was prepared from 10 portions of earthworms according to the method in Example 1; (5) Mix the dry paste obtained in step (3) with earthworm extract, crush and sieve, add starch, and fill into capsules to obtain the final product.

[0127] Example 14: A capsule formulation and its preparation method The preparation method is as follows: (1) Weigh 20 parts of processed Polygonum multiflorum, crush it into coarse particles with a diameter of 8 mm, and extract it twice by heating and reflux with 90% ethanol. The amount of ethanol used in the first extraction is 10 times the amount of medicinal material, and the extraction time is 1 hour. The amount of ethanol used in the second extraction is 10 times the amount of medicinal material, and the extraction time is 1 hour. Combine the filtrates, recover the ethanol under reduced pressure, and concentrate it to a paste with a relative density of 1.0 at 50℃. (2) Add 25 parts of lotus leaf, 10 parts of Cistanche deserticola and 20 parts of Echinops latifolius to water and decoct twice. The first time, add 8 times the amount of water and decoct for 3 hours. The second time, add 6 times the amount of water and decoct for 2 hours. Combine the decoctions and concentrate them to a relative density of 1.0 (50℃). After cooling, add ethanol to make the alcohol content reach 55%. Let stand for 24 hours, filter, recover the ethanol from the filtrate, and concentrate under reduced pressure to a clear extract with a relative density of 1.0 (50℃). (3) Combine the clear pastes obtained in steps (1) and (2), and vacuum dry them to make a dry paste; (4) Earthworm extract was prepared from 25 portions of earthworms according to the method in Example 1; (5) Mix the dry paste obtained in step (3) with earthworm extract, crush and sieve, add starch, and fill into capsules to obtain the final product.

[0128] Example 15: A capsule formulation and its preparation method The preparation method is as follows: (1) Weigh 50 parts of processed Polygonum multiflorum, crush it into coarse particles with a diameter of 50 mm, and extract it twice by heating and reflux with 60% ethanol. The amount of ethanol used in the first extraction is 6 times the amount of medicinal material and the extraction time is 2 hours. The amount of ethanol used in the second extraction is 8 times the amount of medicinal material and the extraction time is 3 hours. Combine the filtrates, recover the ethanol under reduced pressure, and concentrate it to a paste with a relative density of 1.5 at 80℃. (2) Add 10 parts of lotus leaf, 30 parts of Cistanche deserticola and 2 parts of Echinops latifolius to water and decoct twice. The first time, use 15 times the amount of water and decoct for 2 hours. The second time, use 12 times the amount of water and decoct for 3 hours. Combine the decoctions and concentrate them to a relative density of 1.3 (80℃). After cooling, add ethanol to make the alcohol content reach 70%. Let stand for 8 hours, filter, recover the ethanol from the filtrate, and concentrate under reduced pressure to a clear extract with a relative density of 1.5 (80℃). (3) Combine the clear pastes obtained in steps (1) and (2), and vacuum dry them to make a dry paste; (4) Earthworm extract was prepared from 8 portions of earthworms according to the method in Example 1; (5) Mix the dry paste obtained in step (3) with earthworm extract, crush and sieve, add starch, and fill into capsules to obtain the final product.

[0129] Comparative Example 9: A capsule formulation and its preparation method The preparation method is as follows: (1) Weigh 35 parts of processed Polygonum multiflorum, crush it into coarse particles with a diameter of 30 mm, and extract it twice by heating and reflux with 75% ethanol. The amount of ethanol used in the first extraction is 8 times the amount of medicinal material and the extraction time is 2 hours. The amount of ethanol used in the second extraction is 6 times the amount of medicinal material and the extraction time is 2 hours. Combine the filtrates, recover the ethanol under reduced pressure, and concentrate it to a paste with a relative density of 1.3 at 60℃. (2) Add 25 parts of lotus leaf, 15 parts of Cistanche deserticola and 5 parts of Echinops latifolius to water and decoct twice. The first time, add 10 times the amount of water and decoct for 2 hours. The second time, add 8 times the amount of water and decoct for 2 hours. Combine the decoctions and concentrate them to a relative density of 1.3 (60℃). After cooling, add ethanol to make the alcohol content reach 40%. Let stand for 12 hours, filter, recover the ethanol from the filtrate, and concentrate under reduced pressure to a clear extract with a relative density of 1.30 (60℃). (3) Combine the clear pastes obtained in steps (1) and (2), and vacuum dry them to make a dry paste; (4) Earthworm extract was prepared from 10 portions of earthworm using the method described in Comparative Example 1; (5) Mix the dry paste obtained in step (3) with earthworm extract, crush and sieve, add starch, and fill into capsules to obtain the final product.

[0130] Comparative Example 10: A capsule formulation and its preparation method The preparation method is as follows: (1) Weigh 35 parts of processed Polygonum multiflorum, crush it into coarse particles with a diameter of 30 mm, and extract it twice by heating and reflux with 75% ethanol. The amount of ethanol used in the first extraction is 8 times the amount of medicinal material and the extraction time is 2 hours. The amount of ethanol used in the second extraction is 6 times the amount of medicinal material and the extraction time is 2 hours. Combine the filtrates, recover the ethanol under reduced pressure, and concentrate it to a paste with a relative density of 1.3 at 60℃. (2) Add 25 parts of lotus leaf, 15 parts of Cistanche deserticola and 5 parts of Echinops latifolius to water and decoct twice. The first time, add 10 times the amount of water and decoct for 2 hours. The second time, add 8 times the amount of water and decoct for 2 hours. Combine the decoctions and concentrate them to a relative density of 1.3 (60℃). After cooling, add ethanol to make the alcohol content reach 40%. Let stand for 12 hours, filter, recover the ethanol from the filtrate, and concentrate under reduced pressure to a clear extract with a relative density of 1.30 (60℃). (3) Combine the clear pastes obtained in steps (1) and (2), and vacuum dry them to make a dry paste; (4) Earthworm extract was prepared from 10 parts of earthworm according to the method of Comparative Example 2; (5) Mix the dry paste obtained in step (3) with earthworm extract, crush and sieve, add starch, and fill into capsules to obtain the final product.

[0131] Example 3: Quantitative determination of aprotinin in capsule formulations Preparation of reference solution: Weigh an appropriate amount of aprotinin accurately, add 2% acetonitrile and 98% aqueous solution containing 0.15% formic acid to prepare a solution of 200 µg / ml.

[0132] Preparation of the test solution: Accurately weigh 2.5 g of the contents of the capsule formulation prepared in Example 13, add phosphate buffer, sonicate, centrifuge, collect the supernatant, add a small amount of acetone, let stand, centrifuge, wash the precipitate with a small amount of acetone, filter, and dry. Accurately weigh approximately 1 g of the contents of three parallel batches of the formulation, place them in a 10 ml volumetric flask, add 2% acetonitrile and 98% aqueous solution containing 0.15% formic acid to the mark, weigh, sonicate for 10 min, cool, replenish the lost weight with 2% acetonitrile and 98% aqueous solution containing 0.15% formic acid, shake well, and filter through a 0.22 µm microporous membrane to obtain the test solution.

[0133] Column: SHIMSEN Ankylo SEC-150 (5µm, 7.8 x 300 mm); Mobile phase: 2% acetonitrile and 98% aqueous solution containing 0.15% formic acid; Flow rate: 0.6 mL / min; Detection wavelength: 214 nm; Injection volume: 5 µL; Assay method: Precision pipette 5 µL each of the above reference substance solution and test solution, inject into the liquid chromatograph, determine, and you will get the result.

[0134] The detection results are shown in Table 6 below.

[0135] Table 6 Quantitative determination results of aprotinin in the purified capsule preparation

[0136] Effect Example 4 Pharmacodynamic study of the capsule preparation on rats with vascular dementia (VD) I. Materials and methods 1.1 Animals and reagents 60 SPF-grade male SD rats with a body weight of 200 - 240 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the certificate number SCXK (Beijing) 2016 - 0011. They were kept in an SPF-grade environment, and the rats were fed with maintenance feed freely and had free access to water.

[0137] Detection kits for the activities of superoxide dismutase (SOD, batch number A001 - 2 - 2), catalase (CAT, batch number A006 - 1 - 1), and the content of malondialdehyde (MDA, batch number A0030 - 2), Nanjing Jiancheng Bioengineering Institute; Bicinchoninic acid (BCA, batch number JN7462) detection kit, Shanghai Jining Industry Co., Ltd.; Tumor necrosis factor α (TNF-α, batch number H052 - 1 - 2) kit, Nanjing Jiancheng Bioengineering Institute; Interleukin-1β (IL-1β, batch number ml106733) kit, Shanghai Enzyme-linked Biology; Interleukin-6 (IL-6, batch number H007 - 1 - 1) kit, purchased from Nanjing Jiancheng Bioengineering Institute.

[0138] 1.2 Animal model establishment and grouping After one week of acclimatization, SD rats were randomly divided into 6 groups: a sham-operated control group, a VD model group, Example 13 group, comparative examples 9-10 groups, and donepezil hydrochloride group, with 10 rats in each group. The VD animal model was established using the 2-VO method. Rats were fasted for 12 hours and deprived of water for 4 hours before surgery. Anesthesia was administered via intraperitoneal injection of 1% sodium pentobarbital (45 mg / kg). After the rats were fixed in a supine position, their necks were shaved, prepared, and disinfected. An incision was made in the midline of the neck, and the neck muscles were bluntly dissected with sterile cotton balls to expose the bilateral common carotid arteries. The vagus nerve was separated from the common carotid arteries with forceps, and the left and right common carotid arteries were ligated using 3-0 sterile silk sutures. The wounds were sutured to complete the model preparation. Amoxicillin powder was administered to the wounds to prevent infection. In the sham-operated control group, the common carotid artery was only dissected without ligation; other treatments were the same as in the VD model group.

[0139] 1.3 Administration Three days after VD rat modeling, the drug was administered by gavage. The rat dosage was calculated based on the daily clinical dosage for humans. The dosage for groups 13, 9 and 10 was 0.32 g / kg, and the dosage for donepezil hydrochloride group was 0.5 mg / kg. The sham-operated group and VD model group were administered an equal volume of physiological saline by gavage once a day for 30 consecutive days.

[0140] 1.4 Morris Water Maze Experiment The Morris water maze had a diameter of 160 cm, a height of 50 cm, and a depth of 30 cm. The water temperature was maintained at 21±2℃. The circular tank was divided into four quadrants, with four equidistant points marked on the tank wall (N, E, S, W) as the starting points for the experiment. A black circular platform, 12 cm in diameter and 29 cm high, was placed in the center of the fourth quadrant as the analysis area. The platform was submerged 1 cm below the surface. The orientation and navigation experiment was conducted from day 1 to day 5, with training twice a day at least 2 hours apart. In the first training session, rats were placed into the water facing the tank wall from the opposite side of the platform diagonally. In the second training session, rats were randomly placed into the water from either quadrant I or III. During each training session, the rat was stopped if it found the platform within 90 seconds. If the time exceeded 90 seconds, the animal was guided to the platform and remained there for 5 seconds. After each training session, the animal was removed and placed in dry bedding. The instrument automatically recorded the time it took for the rat to find the platform in the water, i.e., the escape latency, which was used as an indicator of learning and memory ability. On day 6, a spatial exploration experiment was conducted. The circular platform in the pool was removed, and rats in each group were placed in the water at the same location in the water maze. The number of times the rats crossed the target area of ​​the platform within 90 seconds and the time required to enter the target quadrant for the first time were recorded.

[0141] 1.5 Effects on oxidative stress levels in rat hippocampal tissue Ten rats were used in each group. Blood was collected from the apex of the heart to prepare serum. After brain harvesting, the hippocampus and other parts were separated. Both serum and hippocampal tissue were stored at -80 ℃. Rats' hippocampal tissue was collected and measured according to the ratio of mass (g):volume (mL). Add homogenizing medium at a ratio of 1:9, homogenize in an ice-water bath, and the supernatant after centrifugation is the hippocampal tissue homogenate; oxidative stress indicators SOD, CAT activity and MDA content are detected by colorimetric method, and the relevant procedures are in accordance with the kit instructions.

[0142] 1.6 Effects on inflammatory factors in rats The contents of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in rat hippocampal tissue were measured by enzyme-linked immunosorbent assay (ELISA).

[0143] 1.7 Small animal MRI scan of brain structure (1) Small animal magnetic resonance brain tissue scans were performed on rats in the sham surgery group, VD model group, and Example 13 group at baseline and after the end of medication. (2) MRI scanner: BRUKER 7T Pharmascan system; (3) Scanning coil: an integrated quadrature transmitting and receiving coil; (4) Scanning parameters: Diffusion tensor imaging (DTI) uses a single-shot excitation spin plane echo sequence: 30 gradient directions, b0 = 0 s / mm2, b = 1000 s / mm2, FOV = 30 mm, matrix = 64×64; field of view (FOV) = 30 mm; slice thickness = 1 mm, gap = 0.2 mm; number of slices = 27. (5) Data acquisition: Rats were gas-anesthetized, and heart rate and respiration were monitored during the scanning process. Each rat underwent MRI examination at the corresponding time points, and T2 high-resolution structural images, DTI, and BOLD fMRI were acquired sequentially (the analysis of T2 and BOLD images is in progress). (6) DTI data preprocessing The data was preprocessed using DSIstudio software. First, the raw BRUKER data was imported into DSIstudio for eddy current correction. The DTI tensor was then solved using a multivariate linear fitting algorithm to obtain B0, FA, and MD maps. The scalp tissue was manually removed, leaving only the brain tissue for the next step of analysis. (7) Analysis method based on whole brain voxels 1) Constructing a population-specific template: First, select a representative B0 image of a sham-operated rat as a reference image, register the B0 images of all other rats onto the reference image through affine transformation, and average all normalized images to create a population-specific template; VBA analysis of FA diagrams: The B0 diagram of each rat was registered to the population-specific template again by affine transformation. For the same rat, the same transformation matrix was used to spatially standardize the FA. The spatially normalized FA images were smoothed using a 6 mm FWHM Gaussian kernel to reduce registration errors during the spatial normalization process. A whole-brain voxel-level statistical analysis was performed on the processed FA images using the SPM12 (SPM12, http: / / www.fil.ion.ucl.ac.uk / spm / ) all-factor model. Repeated measures ANOVA was used to calculate the brain regions with significant group-time point interactions, and the mean FA values ​​of these clusters were extracted. The same processing was performed on the MD images.

[0144] Statistical analysis Statistical analysis was performed using SPSS 20.0 software. All data are presented as mean ± standard deviation. Mean ± s) indicates the statistical significance of data. One-way ANOVA and nonparametric tests were used for continuous data. FA and MD values ​​of significantly interacting brain regions were extracted at time points (baseline, post-treatment) from the group (VD model group, Example 13 group) and analyzed using SPSS 20.0 software. Repeated measures ANOVA was used, and pairwise comparisons between groups at each time point were performed using multivariate ANOVA. The least significant difference (LSD) test was used, and p < 0.05 was considered statistically significant.

[0145] 2. Results 2.1 Effects of capsule formulation on cognitive function in VD rats The results of the Morris water maze behavioral analysis are shown in the table below.

[0146] Table 7 Escape Incubation Period

[0147] Note: Compared with the sham surgery group, **P<0.01; compared with the model group, ##P<0.01; compared with Example 13, aP<0.05, aaP<0.01.

[0148] Table 8. Time of arrival in quadrant and number of crossings

[0149] Note: Compared with the sham surgery group, **P<0.01; compared with the model group, ##P<0.01; compared with Example 13, aP<0.05, aaP<0.01.

[0150] Experimental results showed that the escape latency of all groups decreased from day 1 to day 5. Compared with the VD model group, the escape latency of the sham-operated control group and the Example 13 group was significantly shorter on days 3, 4, and 5 (P<0.05). Spatial exploration results showed that compared with the sham-operated control group, the VD model group had a significantly longer time to reach the target quadrant (P<0.01) and a significantly fewer number of crossings (P<0.01). Compared with the VD model group, the Example 13 group had a significantly shorter time to reach the target quadrant (P<0.01) and a significantly more number of crossings (P<0.01). These behavioral results indicate that the capsule formulation of the present invention can significantly improve the learning and memory abilities of the VD rat model.

[0151] 2.2 Effects of capsule formulation on oxidative stress levels and inflammatory factors in hippocampal tissue of VD rats The results of the analysis of oxidative stress levels and inflammatory factors in the hippocampus of VD rats are shown in the table below.

[0152] Table 9. Effects of the composition on oxidative stress levels in rat hippocampus (mean±SD, n=10)

[0153] Note: Compared with the sham surgery group, **P<0.01; compared with the model group, ##P<0.01; compared with Example 13, aaP<0.01.

[0154] As shown in the table above, compared with the sham-operated group, the levels of CAT and SOD in the hippocampus of rats in the model group were significantly decreased (P < 0.01), while the level of MDA was significantly increased (P < 0.01), indicating that the antioxidant capacity of rats with vascular dementia was significantly reduced. Compared with the model group, the levels of CAT and SOD in the hippocampus of rats in Example 13 group and the positive drug group were significantly increased (P < 0.01), while the level of MDA was significantly decreased (P < 0.01). Compared with the model group, there were no significant changes in the SOD and CAT activities and MDA content in the hippocampus of rats in Comparative Examples 9-10 groups, with no statistically significant differences. Comparative Examples 9-10 groups showed statistically significant differences compared with Example 13 group (P < 0.01), indicating that the earthworm composition containing the improved earthworm preparation process had better effects.

[0155] Table 10 Effects of the composition on the levels of inflammatory factors in rats (mean±SD, n=10)

[0156] Note: Compared with the sham surgery group, **P<0.01; compared with the model group, ##P<0.01; compared with Example 13, aaP<0.01.

[0157] As shown in the table above, compared with the sham-operated group, the levels of TNF-α, IL-1β, and IL-6 in the hippocampus of the model group rats were significantly increased (P < 0.01), indicating increased inflammation levels and aggravated hippocampal damage in the vascular dementia model rats. After 30 days of administration, compared with the model group, the levels of TNF-α, IL-1β, and IL-6 in the positive drug group and Example 13 group were significantly decreased (P < 0.01). The effect of Example 13 group was comparable to that of the positive drug group, suggesting that the composition can significantly inhibit the inflammatory response in vascular dementia rats and improve hippocampal tissue. Compared with the model group, there was no significant improvement in the levels of TNF-α, IL-1β, and IL-6 in Comparative Example 9-10 groups. Comparative Example 9-10 groups showed statistically significant differences compared with Example 13 group (P < 0.01), indicating that the earthworm composition obtained by the improved earthworm preparation process had better effects.

[0158] 2.3 Whole-brain voxel analysis results of VD rats treated with capsule formulation As shown by whole-brain voxel analysis, the interaction between the sham-operated control group, the VD model group, and the capsule formulation group of Example 13 and the time point was significant in the following brain regions (P<0.005): bilateral caudate nuclei (Left Caudate1, Right Caudate2), right thalamus, and right hippocampus (see...). Figure 4 No significant interaction was found in the MD values. This indicates that there are significant differences in the dynamic evolution of nerve fiber integrity over time among the sham-operated control group, the VD model group, and the implementation group.

[0159] 2.4 Protective effect of capsule formulation on the integrity of nerve fiber bundles in VD rats Further analysis was conducted by extracting the average FA values ​​from the above clusters. Pairwise comparisons showed that, at baseline, the FA values ​​of the bilateral caudate nuclei (Left Caudate1, Right Caudate2) and right hippocampus in the capsule formulation group were significantly lower than those in the VD model group. However, after medication, the FA values ​​of the bilateral caudate nuclei, right thalamus, and right hippocampus in the capsule formulation group were all significantly higher than those in the VD model group (see...). Figure 5The results indicate that the pharmaceutical composition of the present invention has a significant protective effect on the integrity of white matter fibers in the above-mentioned brain regions. Decreased FA values ​​in the brain regions of VD rats reflect a reduction in neuronal fibers, widespread demyelination, and decreased white matter fiber integrity in the brain tissue. Whole-brain voxel analysis results show that the capsule formulation of the present invention has a certain repair effect on nerve fiber damage in the bilateral caudate nucleus and right thalamus of VD rats.

[0160] Example 5: Clinical study of capsule formulation for schizophrenia A multicenter case registry study was conducted on the capsule formulation prepared in Example 13 of the present invention to evaluate the efficacy and safety of the capsule formulation of the present invention in treating patients with schizophrenia. A total of 10 subjects who met the inclusion criteria were included, including 8 females and 2 males, with the youngest age being 18 years old, the oldest age being 60 years old, and the mean age being 35.2 ± 11.57 years.

[0161] I. Research Subjects 1. Diagnostic criteria: (1) Meets the diagnostic criteria for schizophrenia in the International Classification of Diseases, Tenth Edition (ICD-10), with a total score of 60 or above on the Positive and Negative Syndrome Scale (PANSS). (2) Diagnostic criteria for TCM syndrome (liver and kidney deficiency with phlegm and blood stasis obstructing the collaterals); (3) Screening indicators: Mini-Mental State Examination (MMSE); 2. Inclusion criteria: (1) Meets the diagnostic criteria for schizophrenia in the International Classification of Diseases, Tenth Edition (ICD-10), with a total score of 60 or above on the Positive and Negative Syndrome Scale (PANSS). (2) The Mini-Mental State Examination (MMSE) score is between 10 and 26. (3) Diagnostic criteria for TCM syndrome (liver and kidney deficiency with phlegm and blood stasis obstructing the collaterals); (4) The total course of disease is less than three years, and the patient has not used related drugs before enrollment, or has stopped taking the drugs for at least 2 months; (5) Age between 18 and 60 years, no history of electroconvulsive therapy, and no history of head trauma. (6) The patient and his / her legal guardian voluntarily agree to participate in this study and sign the informed consent form. 3. Exclusion criteria: (1) Individuals with a known allergy to the test drug or any of its components; (2) History of physical diseases such as heart, liver, kidney and gastrointestinal diseases or serious systemic diseases such as immune and endocrine metabolic disorders; (3) Pregnant or breastfeeding women; (4) Those with a history of drug or psychoactive substance abuse; (5) Researchers who believe that they are not suitable to participate in the registration for other reasons. 6. Termination Criteria: (1) If serious adverse reactions occur during the study, and the subject or researcher decides to terminate the study; (2) All research subjects who have filled out informed consent forms and passed the screening to enter the study, or whose legal guardians do not wish to continue during the clinical research process and request to withdraw from the study, or who withdraw at any time and for any reason.

[0162] II. Evaluation Indicators 1. Main therapeutic indicators: Mini-Mental State Examination (MMSE) Record time points: Day of enrollment, 90±7 days of medication, and 180±7 days of medication. 2. Secondary therapeutic indicators (1) Panss scale evaluation Record time points: Day of enrollment, 90±7 days of medication, and 180±7 days of medication. (2) Traditional Chinese Medicine Syndrome Diagnosis Table Record time points: Day of enrollment, 90±7 days of medication, and 180±7 days of medication. III. Results 1. Therapeutic effect analysis Table 11 Comparison of total scores on each scale before treatment and at 90 and 180 days after treatment.

[0163] Note: Compared with the initial group, #P<0.05, ##P<0.01; compared with the initial group, **P<0.01; compared with visit 1, aP<0.05, aaP<0.01.

[0164] The results showed that with the extension of treatment time, the scores of each scale showed significant dynamic improvement: MMSE score: significantly higher at 90 days of treatment than at the start of treatment (P<0.01), and further higher at 180 days of treatment, with extremely significant differences compared with the start of treatment and at 90 days of treatment (P<0.01), indicating that the cognitive function caused by schizophrenia in patients continued to improve with the treatment process, and demonstrating that the drug of the present invention has a beneficial effect on cognitive impairment caused by schizophrenia; PANSS score: Significantly lower than the baseline score at 90 days of treatment (P<0.01), and further lower at 180 days of treatment. The difference was extremely significant compared with the baseline score and at 90 days of treatment (P<0.01), indicating that the positive and negative symptoms of schizophrenia and the mental state of patients continued to improve with the progress of treatment. The scores on the Traditional Chinese Medicine (TCM) syndrome diagnosis scale were significantly lower at 90 days of treatment compared to the initial group (P<0.05), and further decreased at 180 days of treatment. There were extremely significant differences compared to the initial group (P<0.01) and significant differences compared to the initial group at 90 days of treatment (P<0.05). This suggests that the TCM syndrome manifestations continued to lessen with the progress of treatment, indicating that the drug of this invention has excellent therapeutic effects on schizophrenia.

[0165] In summary, the drug of the present invention has a clear therapeutic effect on schizophrenia and cognitive impairment caused by schizophrenia, and the mental symptoms and cognitive function, including negative and positive symptoms, show a continuous improvement trend with the extension of treatment time.

[0166] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. An earthworm extract, characterized in that: The earthworm extract comprises component A with a molecular weight <10kDa and component B with a molecular weight of 10-66kDa, wherein the distribution ratio of component A to component B is 40%-90%: 10%-60%.

2. The earthworm extract according to claim 1, characterized in that: The earthworm extract contains components A and B in a ratio of 40-65% and 35-60%, respectively.

3. The earthworm extract according to claim 1, characterized in that: The earthworm extract contains components A and B in a ratio of 45-55%:45-55%.

4. A method for preparing earthworm extract, characterized in that: The process includes the following steps: weighing earthworms and crushing them into coarse powder, adding water and stirring to form a homogenate, combining the homogenates, adding ethanol to make the alcohol content 5-25%, and allowing it to stand for a first alcohol precipitation; taking the supernatant, adding ethanol to make the alcohol content 40-65%, and allowing it to stand for a second alcohol precipitation; taking the precipitate, washing it with propanol, and drying it at a temperature of 40℃-70℃ to obtain the earthworm extract; preferably, the drying temperature of the precipitate is 55-65℃.

5. The method for preparing earthworm extract according to claim 4, characterized in that: The single-stage alcohol precipitation involves adding ethanol to achieve an alcohol content of 5-20%; preferably 5-15%. The secondary alcohol precipitation involves adding ethanol to achieve an alcohol content of 40-60%; preferably 45-55%. Optionally, the primary alcohol precipitation temperature is 2-20°C; preferably, the primary alcohol precipitation temperature is 2-15°C; more preferably, it is 2-10°C. Optionally, the secondary alcohol precipitation temperature is 2-20℃, preferably, the secondary alcohol precipitation temperature is 2-15℃, and more preferably 2-10℃; Optionally, the initial alcohol precipitation settling time is 6-24 hours, preferably 8-18 hours, and more preferably 8-15 hours. Optionally, the settling time for the secondary alcohol precipitation is 6-24 hours, preferably 8-18 hours, and more preferably 8-15 hours.

6. An earthworm extract, characterized in that, The earthworm extract was prepared according to the method described in claim 4 or claim 5.

7. A traditional Chinese medicine extract composition, characterized in that: The raw materials of the extraction composition consist of the following medicinal ingredients: 20-50 parts of processed Polygonum multiflorum, 10-30 parts of lotus leaf, 10-30 parts of Cistanche deserticola, 2-20 parts of Echinops latifolius, and 8-25 parts of earthworm; wherein the earthworm extract is the earthworm extract according to any one of claims 1-3 or prepared by the preparation method according to claim 4 or 5.

8. The traditional Chinese medicine extraction composition according to claim 7, characterized in that: The preparation method of the traditional Chinese medicine composition includes the following steps: (1) Weigh the prepared Polygonum multiflorum, crush it, extract it by heating and reflux with ethanol, combine the filtrates, recover the ethanol under reduced pressure, and concentrate it into a paste; (2) Boil lotus leaves, Cistanche deserticola and Echinops latifolius in water, combine the decoctions, concentrate the decoction to a relative density of 1.0-1.3 at 50-80℃, cool and add ethanol, let stand, filter, recover the ethanol from the filtrate, and concentrate under reduced pressure to a clear extract with a relative density of 1.0-1.5 at 50-80℃. (2) Combine the clear pastes obtained in steps (1) and (2), and vacuum dry them to make a dry paste; (4) The dry extract obtained in step (3) is mixed with the earthworm extract according to any one of claims 1-3 or the earthworm extract prepared by the preparation method according to claim 4 or 5 to obtain the traditional Chinese medicine extract composition.

9. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the traditional Chinese medicine extract composition as described in claim 7 or 8 and pharmaceutically acceptable excipients, wherein the pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, lubricants, disintegrants, flow aids, and wetting agents; and the dosage form of the pharmaceutical preparation includes, but is not limited to, granules, tablets, powders, capsules, oral liquids, injections, sprays, aerosols, powder inhalers, eye drops, nasal drops, suppositories, pills, microspheres, or tinctures.

10. The pharmaceutical preparation according to claim 9, characterized in that, The pharmaceutical preparation is a capsule preparation, and further, the aprotinin content in the capsule preparation is not less than 0.4 mg / g.

11. The use of the traditional Chinese medicine extract composition according to claim 7 or 8 or the pharmaceutical preparation according to claim 9 or 10 in the preparation of a drug for treating schizophrenia, characterized in that: Schizophrenia includes one or more of the following: negative symptoms, positive symptoms, and cognitive impairment.

12. The use of the traditional Chinese medicine extract composition according to claim 7 or 8 or the pharmaceutical preparation according to claim 9 or 10 in the preparation of a drug for treating cognitive impairment, characterized in that: The cognitive impairment disease mentioned is selected from one or more of Alzheimer's disease, vascular dementia, mild cognitive impairment, frontotemporal dementia, Lewy body dementia, Parkinson's disease dementia, and other types of dementia.

13. The use of the traditional Chinese medicine extract composition according to claim 7 or 8 or the pharmaceutical preparation according to claim 9 or 10 in the preparation of a drug for treating cognitive impairment in schizophrenia, characterized in that: The cognitive impairments mentioned include, but are not limited to, attention deficit disorder, impaired working memory, slowed information processing speed, and learning and memory disorders.

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