Formula for improving thrombus dissolving capacity of human body, preparation method and application thereof

Abstract

The invention provides a composition for improving the thrombolysis capacity of a human body, a preparation method and application thereof.The composition is prepared from, by weight, 5-30 parts of earthworm protein, 30-120 parts of hippophae rhamnoides total flavone extract, 50-150 parts of silymarin extract, 20-80 parts of maltodextrin and 15-60 parts of pregelatinized starch, the earthworm protein activates a fibrinolytic system to promote t-PA to release and dissolve thrombus, and the hippophae rhamnoides total flavone extract activates the fibrinolytic system to promote t-PA to release and dissolve thrombus; the hippophae rhamnoides total flavonoids regulate the ratio of PGI2 / TXA2 to inhibit platelet aggregation and reduce blood fat, the silymarin protects the liver function to enhance LDL-C metabolism clearing, the hippophae rhamnoides total flavonoids, the silymarin and the hippophae rhamnoides total flavonoids cooperate to form a thrombolysis-antithrombosis-metabolism regulation three-dimensional intervention system, a whole-course low-temperature mixing direct tabletting process is adopted

Description

Technical Field

[0001] This invention belongs to the field of natural drug composition technology, specifically relating to a formulation, preparation method and application of a method for enhancing the body's own ability to dissolve blood clots. Background Technology

[0002] Cardiovascular and cerebrovascular diseases are the leading cause of death worldwide, and their core pathological mechanism lies in the formation of intravascular thrombi and microcirculatory disturbances. The thrombus formation process involves three key steps: activation of the coagulation cascade, abnormal platelet aggregation, and impaired fibrinolytic system function. Under normal physiological conditions, the body's endogenous fibrinolytic system converts plasminogen into plasmin via tissue plasminogen activator (t-PA), which then degrades fibrin clots to maintain vascular patency. However, with age, metabolic disorders, and increased vascular endothelial damage, the release capacity of t-PA significantly decreases, while the activity of plasma plasminogen activator inhibitor-1 (PAI-1) abnormally increases, leading to an imbalance in the fibrinolytic system and a significant reduction in thrombus clearance capacity.

[0003] Statistics show that the direct causes of ischemic stroke and acute myocardial infarction are closely related to the formation or detachment of arterial thrombi. Epidemiological studies indicate that hyperlipidemia, hypertension, and diabetes are the three major independent risk factors for thrombosis, and the prevalence of these three metabolic diseases is showing a continuous upward trend, making the prevention and control of thrombosis-related diseases increasingly challenging. Microcirculatory disturbances, as a common pathological basis for many chronic diseases, are also closely related to the formation of microthrombi in microvessels and vascular endothelial damage. Therefore, developing safe, mild, and long-term usable natural functional formulations by improving the body's own thrombolytic capacity has significant public health implications for both primary and secondary prevention of cardiovascular and cerebrovascular diseases.

[0004] Currently used thrombolytic drugs mainly include urokinase, streptokinase, and recombinant tissue plasminogen activator (rt-PA). Although these drugs can directly dissolve blood clots, they have limitations such as high bleeding risk, narrow therapeutic window, high cost, and the need for strict medical monitoring, making them unsuitable as a means of long-term prevention and chronic thrombosis management. In the field of traditional Chinese medicine, blood-activating and stasis-removing formulas such as Xuefu Zhuyu Decoction and Buyang Huanwu Decoction have a long history of application, but most lack modern pharmacological mechanism explanations and precise quality control standards. Their formulations are complex, and the interactions between their components are unclear, making it difficult to meet the requirements of modern drug registration and approval.

[0005] In recent years, research on functional compositions based on natural active proteins and plant extracts has received widespread attention. Earthworm protein is a mixture of active proteins extracted from earthworms, and studies have confirmed its pharmacological activities in promoting fibrinolysis and improving microcirculation. However, existing earthworm protein products generally suffer from large batch-to-batch quality fluctuations, unclear active ingredients, and slow onset of action when used alone. Among published patent documents, CN116270984A discloses a composition for improving vascular embolism containing earthworm protein peptides, but it relies on traditional Chinese medicine extracts for promoting blood circulation and removing blood stasis (such as Angelica sinensis and Ligusticum chuanxiong) as auxiliary components and does not address the effects of flavonoids on platelet aggregation inhibition and lipid metabolism regulation. CN115607657A discloses a composition of nattokinase and earthworm protein peptides, but this scheme only involves the superposition of direct fibrinolytic enzyme activity and lacks intervention on upstream links of thrombus formation (dyslipidemia, vascular endothelial damage, and excessive platelet activation), thus failing to achieve a synergistic effect of preventing thrombus formation and promoting thrombolysis.

[0006] Therefore, how to design a safe, effective, and multi-target synergistic natural formula that can comprehensively improve the body's own thrombolytic ability, activate the endogenous fibrinolytic system, and inhibit upstream factors of thrombus formation is a technical problem that urgently needs to be solved in this field.

[0007] From a pharmacological perspective, an ideal thrombolytic formulation should simultaneously target three levels: fibrinolytic system activation, antiplatelet aggregation, and lipid metabolism regulation. While direct thrombolytic strategies relying solely on plasmin or plasminogen activator can degrade existing fibrin clots, they cannot prevent the continuous formation of new thrombi or eliminate metabolic risk factors leading to recurrent thrombus formation (such as hypercholesterolemia and hypertriglyceridemia). Studies have shown that a 1 mmol / L decrease in plasma low-density lipoprotein cholesterol levels can reduce the risk of cardiovascular events by approximately 22%. Furthermore, the dynamic balance between thromboxane A2 receptors and prostacyclin receptors on the platelet surface is a key factor determining platelet aggregation; a decreased PGI2 / TXA2 ratio is closely related to the occurrence of atherosclerosis and coronary heart disease. Therefore, developing a natural formulation that simultaneously exerts effects across the three dimensions of fibrinolytic activation, antiplatelet aggregation, and hepatic lipid metabolism regulation has significant clinical application value and industrialization prospects. Summary of the Invention

[0008] The purpose of this invention is to provide a formulation and preparation method for improving the body's own thrombolytic ability. This formulation, through the synergistic combination of three core active components—earthworm protein, total flavonoids from sea buckthorn, and silymarin extract—forms a three-dimensional targeted intervention at three levels: activating the fibrinolytic system, inhibiting thrombus formation, and promoting hepatic lipid metabolism. This overcomes the technical defects of existing technologies, such as incomplete single thrombolytic mechanisms, slow onset of action, and insufficient safety.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A formula for enhancing the body's own thrombolytic ability comprises, by weight, the following components: 5-30 parts earthworm protein, 30-120 parts total flavonoid extract from sea buckthorn, 50-150 parts silymarin extract, 20-80 parts maltodextrin, and 15-60 parts pregelatinized starch. The earthworm protein contains at least 15% fibrinolytic system activating protein of its total protein content, the total flavonoid extract from sea buckthorn contains at least 60% total flavonoids, and the silymarin extract contains at least 50% silymarin.

[0011] The preparation method of the above-mentioned formulation includes the following steps: Step 1, mixing silymarin extract and pregelatinized starch evenly at 25±5℃ and stirring for 3-8 min; Step 2, adding total flavonoid extract of sea buckthorn and maltodextrin to the mixture obtained in Step 1 and stirring for 8-15 min at 25±5℃; Step 3, adding earthworm protein to the mixture obtained in Step 2 and stirring at low speed for 12-20 min at 20±3℃; Step 4, passing the material obtained in Step 3 through a 16-24 mesh sieve, mixing evenly, and directly compressing into tablets, with the tableting pressure controlled at 5-15 kN and the tablet weight at 400-600 mg.

[0012] The beneficial effects of this invention are as follows:

[0013] First, the formulation of this invention establishes a three-dimensional targeted synergistic system of "fibrinolysis activation-antithrombosis-metabolic regulation". The fibrinolysis system activating protein in earthworm protein promotes the release of t-PA from vascular endothelial cells, thereby activating plasminogen to plasmin, which directly degrades fibrin to achieve thrombolysis. Total flavonoids from sea buckthorn inhibit platelet aggregation and thrombus formation by increasing the ratio of prostacyclin (PGI2) to thromboxane A2 (TXA2), while simultaneously reducing plasma total cholesterol and triglyceride levels, thus reducing the formation of atherosclerotic plaques at the source. Silymarin enhances the liver's metabolic clearance of low-density lipoprotein cholesterol by protecting hepatocyte membrane integrity and promoting liver protein synthesis, improving abnormal lipid profiles and reducing the risk of thrombosis at the metabolic level. The synergistic effect of these three components forms a complete intervention chain from thrombolysis and thrombus prevention to lipid metabolism optimization, which has significant advantages over single thrombolytic components or simple two-component combinations.

[0014] The pharmacokinetic characteristics of silymarin are instructive for formulation design. After oral administration, silymarin is primarily absorbed in the duodenum and upper jejunum, reaching peak plasma concentration in approximately 2–4 hours, with a half-life of approximately 6–8 hours. It is excreted via bile in the form of glucuronide and sulfate conjugates, exhibiting a significant enterohepatic circulation effect. This characteristic allows silymarin to maintain a high local concentration in the liver, which is beneficial for its hepatoprotective and lipid metabolism-promoting pharmacological effects. The combination of silymarin and total flavonoids from sea buckthorn in this invention is particularly significant: quercetin in total flavonoids from sea buckthorn has been shown to inhibit the efflux of P-glycoprotein, thereby increasing the retention time and transmembrane transport efficiency of silymarin in intestinal epithelial cells, resulting in an oral bioavailability of silymarin that is approximately 25%–40% higher than when administered alone. This synergistic effect at the pharmacokinetic level is another important scientific basis for the three-component formulation design of this invention.

[0015] Secondly, the present invention adopts low-temperature operation and direct tableting process throughout the process, which maximizes the preservation of the enzymatic activity of earthworm protein and the bioactivity of total flavonoids from sea buckthorn. The preparation process is simple and efficient, and is suitable for industrial production.

[0016] Third, all components of the formulation of this invention are of natural origin, have high safety, and have no obvious toxic side effects with long-term use. They are suitable for daily prevention of thrombosis in high-risk groups and for adjuvant intervention during the thrombosis recovery period. Attached Figure Description

[0017] Figure 1 This is a comparison diagram of the fibrin plate melting zone area of ​​Example 1 and Comparative Examples 1-3 of the present invention.

[0018] Figure 2 This is a comparison of the wet weight of rat common carotid artery thrombi in Example 1 of the present invention and Comparative Examples 1-3.

[0019] Figure 3 This is a comparison chart of four blood lipid indicators in rats in Example 1 of the present invention and Comparative Examples 1-3.

[0020] Figure 4 This is a comparison chart of rat plasma t-PA activity and PAI-1 activity between Example 1 and Comparative Examples 1-3 of the present invention.

[0021] Figure 5 This is a comparison chart of the PGI2 / TXA2 ratio in rat plasma of Example 1 and Comparative Examples 1-3 of the present invention.

[0022] Figure 6A HE-stained pathological section of common carotid artery tissue from model group rats (×400), scale bar = 50μm.

[0023] Figure 6BThe image shows a HE-stained pathological section of the common carotid artery tissue from Comparative Example 1 (×400), scale bar = 50 μm.

[0024] Figure 6C The image shows a HE-stained pathological section of the common carotid artery tissue from Comparative Example 3 (×400), scale bar = 50 μm.

[0025] Figure 6D The image shows a HE-stained pathological section of rat common carotid artery tissue from Example 1 (×400), scale bar = 50 μm.

[0026] Figure 7 This is a comparison chart of antioxidant indices in rat plasma and liver tissue of Example 1 and Comparative Examples 1-3 of the present invention. Detailed Implementation

[0027] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are only used to illustrate the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0028] The raw materials used in this invention have the following sources and specifications. Earthworm protein is obtained from fresh Eisenia foetida or Pheretima asp. pergillum through low-temperature water extraction, enzymatic hydrolysis, ultrafiltration separation, and freeze-drying. The fibrinolysis system activating protein content is no less than 15% of the total protein, the protein purity is no less than 85%, and the moisture content is no more than 5%. The fibrinolysis system activating protein is a specific protein obtained by further screening and separation from earthworm protein, with a molecular weight range of 20–35 kDa, capable of specifically activating the t-PA release pathway on the surface of vascular endothelial cells. The extraction process includes: pulverizing dried earthworms through an 80-mesh sieve, adding 8–12 times the mass of deionized water, extracting at 4–10°C for 2–4 hours, centrifuging at 12000 r / min for 20 minutes, collecting the supernatant, fractionating through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 50 kDa sequentially, collecting the 10–50 kDa fraction, and freeze-drying to obtain earthworm protein powder.

[0029] The quality control of earthworm protein was carried out using the following methods: Protein content was determined using the Kjeldahl method (Chinese Pharmacopoeia 2020 General Chapter 0731), with the crude protein content calculated by multiplying the nitrogen content by a conversion factor of 6.25. Identification and content determination of fibrinolytic system activating proteins were performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using molecular weight standards as a reference, the target protein should show clear bands in the 20–35 kDa range. Gray-scale scanning of the bands was performed using a gel imaging system. The proportion of the target band's gray value to the total protein band's gray value represents the fibrinolytic system activating protein content, and this proportion should not be less than 15%. Fibrinolytic activity was determined using the fibrin plate method, with a standard curve established using international urokinase standards. The fibrinolytic activity per gram of earthworm protein powder should not be less than 2000 IU. The identification of earthworm raw materials utilizes DNA barcoding technology, with cytochrome c oxidase subunit I (COI) gene sequence comparison to confirm species origin, ensuring that the raw materials used are *Eisenia fetida* or *Pheretima aspergillum*, excluding adulteration with other non-medicinal earthworm species. Heavy metal and pesticide residues in the raw materials are tested according to the relevant standards for animal medicinal materials in the Chinese Pharmacopoeia.

[0030] The total flavonoid extract of sea buckthorn was prepared by ethanol reflux extraction using the pomace of sea buckthorn (Hippophae rhamnoides L.) as raw material. The specific process is as follows: dried sea buckthorn pomace is pulverized and passed through a 40-mesh sieve, and 6-10 times its volume of 60%-80% ethanol solution is added. The mixture is refluxed at 60-70℃ for 2-3 times, each time for 1.5-2.5 hours. The combined extracts are concentrated under reduced pressure, purified using a macroporous resin (AB-8 type), and then eluted with 30% ethanol to remove impurities, followed by 60%-70% ethanol. The eluent is concentrated under reduced pressure and spray-dried to obtain the total flavonoid extract of sea buckthorn with a total flavonoid content of not less than 60%. The main active components in the total flavonoids of sea buckthorn include four aglycone compounds: isorhamnetin, quercetin, myricetin, and kaempferol. Isorhamnetin inhibits platelet cyclooxygenase activity and reduces TXA2 synthesis; quercetin promotes the synthesis and release of PGI2 by vascular endothelial cells; kaempferol reduces arachidonic acid release by inhibiting phospholipase A2 activity; and myricetin has the ability to directly scavenge superoxide anion free radicals. These four active ingredients synergistically regulate the PGI2 / TXA2 balance, inhibiting thrombus formation from both anti-platelet aggregation and antioxidant perspectives.

[0031] The purification process of total flavonoid extract from sea buckthorn has a decisive impact on the quality of the final product. Before purification with macroporous resin, the extract needs to be concentrated under reduced pressure to a relative density of 1.10–1.15 (measured at 60℃), and then loaded onto an AB-8 macroporous adsorption resin column at a flow rate of 2 BV / h, with the resin volume being 3–5 times the volume of the concentrated extract. After loading, the extract is first washed with 5 BV of purified water to remove polar impurities such as sugars and organic acids, then eluted with 3 BV of 30% ethanol to remove low-polarity pigments and waxes, and finally eluted with 5 BV of 60%–70% ethanol to collect the target flavonoid fraction. The eluent is concentrated under reduced pressure to an appropriate volume in a thin-film evaporator below 50℃ and then spray-dried with an inlet air temperature of 160–180℃, an outlet air temperature of 80–90℃, and a nebulization pressure of 0.2–0.3 MPa, ultimately obtaining a free-flowing, pale yellow powder. This purification process achieves a flavonoid recovery rate of over 85%, has good reproducibility, and is suitable for industrial-scale production. The total flavonoid content of the extract was determined by ultraviolet-visible spectrophotometry, using rutin as a standard, and colorimetric analysis was performed at a wavelength of 510 nm. The monomer content of isorhamnetin, quercetin, myricetin and kaempferol was analyzed by high performance liquid chromatography to ensure that the total content of the four aglycone compounds was not less than 40% of the total flavonoid content.

[0032] Silymarin extract was prepared from milk thistle (Silybummarianum (L.) Gaertn.) seeds. Dried milk thistle seeds were pulverized and passed through a 60-mesh sieve. The extracts were refluxed three times with an acetone-water mixture (7:3 v / v) for 2 hours each time, controlled at 55–65°C. The combined extracts were distilled under reduced pressure to recover acetone. The aqueous phase was extracted three times with ethyl acetate. The combined organic phases were concentrated under reduced pressure, and after adsorption with silica gel, the extract was vacuum dried to obtain silymarin extract, with a silymarin content of not less than 50%. The hepatoprotective mechanism of silymarin includes: stabilizing hepatocyte membranes to prevent the entry of hepatotoxic substances such as carbon tetrachloride into cells; promoting hepatocyte RNA polymerase I activity to restore protein synthesis function; scavenging reactive oxygen species in liver tissue to reduce lipid peroxidation levels; and inhibiting the NF-κB pathway to alleviate inflammatory responses. Improved liver function directly enhances the liver's receptor-mediated endocytosis clearance efficiency of low-density lipoprotein cholesterol in the circulating blood, reduces plasma oxidized low-density lipoprotein levels, and thus reduces the atherosclerotic process caused by its deposition in the vascular endothelium.

[0033] Maltodextrin, used as a filler and stabilizer, has a glucose equivalent of 15–20, providing good flowability during tableting while encapsulating earthworm protein to slow its inactivation rate in the acidic environment of the stomach. Pregelatinized starch, used as a disintegrant and binder, has a pregelatinization degree of 85%–95%. After oral administration, it rapidly swells upon contact with water, promoting tablet disintegration and ensuring the full release and absorption of the active ingredients in the upper small intestine.

[0034] The formulation of this invention follows these principles in terms of component proportions. Earthworm protein, as the core thrombolytic component, is determined based on the dose-response relationship of t-PA activation efficiency: preliminary experiments show that when the amount of earthworm protein is less than 5 parts, the increase in plasma t-PA activity is less than 30%, and the thrombolytic effect is not significant; when the amount exceeds 30 parts, the increase in t-PA activity plateaus, and further increases in amount do not bring additional thrombolytic benefits, and excessively high protein content will affect the tablet's compression molding performance. The dosage range of sea buckthorn total flavonoid extract (30–120 parts) is determined based on the dose-response curve of the PGI2 / TXA2 ratio: below 30 parts, the ratio improvement is not significant; the optimal efficiency range is reached around 75 parts; beyond 120 parts, marginal benefits decrease and gastrointestinal discomfort may occur. The dosage range of silymarin extract (50–150 parts) is determined with reference to its protective effect curve on hepatic alanine aminotransferase; the optimal balance of protective effect on hepatocytes is reached around 100 parts. The amount of maltodextrin and pregelatinized starch used is determined according to the requirements of the tablet forming process. If the amount of maltodextrin is too low, the tablets will have poor flowability and large differences in tablet weight. If the amount of pregelatinized starch is too low, the disintegration time will be too long, affecting drug release.

[0035] This invention employs a direct compression process instead of wet granulation, a choice based on the following technical considerations. Wet granulation requires the use of an aqueous binder solution or ethanol solution for wetting and granulation, followed by drying at 40–60°C until the moisture content is within acceptable limits. This exposure to moisture and temperature during the process leads to a 20%–40% loss of enzyme activity in earthworm protein. Furthermore, the vigorous stirring and extrusion during granulation can cause irreversible damage to the protein's spatial structure. This invention, by selecting maltodextrin and pregelatinized starch, which have good compressibility, as excipients, allows the material to achieve sufficient flowability and compressibility without granulation. Pregelatinized starch exhibits strong binding force at room temperature, replacing the role of the binder in traditional wet granulation; the spherical particle structure of maltodextrin imparts excellent flowability to the material, ensuring that tablet weight variation is controlled within specified limits during compression. Another advantage of direct tableting is its high production efficiency and simple process. It only takes about 45 minutes to 1 hour from raw material mixing to tableting, which shortens the production cycle by about 70% compared to wet granulation, thus helping to reduce production costs and increase capacity.

[0036] The ratio of earthworm protein to total flavonoids from sea buckthorn is crucial for the synergistic effect. When the weight ratio of earthworm protein to total flavonoids from sea buckthorn is between 1:5 and 1:10, the synergistic effect coefficient (measured by a combination of fibrinolytic activity and thrombus weight reduction rate) reaches its highest value. This is because total flavonoids from sea buckthorn, while inhibiting thrombus formation, reduce blood viscosity, which facilitates the penetration and distribution of active proteins from earthworm protein to the thrombus site. When silymarin is also present, the liver's clearance efficiency of oxidized low-density lipoprotein is improved, the degree of oxidative stress exposure of vascular endothelial cells is reduced, and the endothelial cells' ability to synthesize t-PA is restored, further amplifying the fibrinolytic activation effect of earthworm protein. This cascade amplification effect among the three components is the core advantage of this invention, distinguishing it from existing two-component thrombolytic formulations.

[0037] Example 1: Tablets were prepared according to the following formulation. 10 parts earthworm protein, 75 parts total flavonoid extract from sea buckthorn (total flavonoid content 70%), 100 parts silymarin extract (silymarin content 55%), 50 parts maltodextrin, and 40 parts pregelatinized starch were taken. First, 100 parts silymarin extract and 40 parts pregelatinized starch were placed in a V-type mixer and mixed at 20 rpm for 5 minutes at room temperature (25°C). Then, 75 parts total flavonoid extract from sea buckthorn and 50 parts maltodextrin were added, and the mixture was stirred at 20 rpm for 10 minutes while maintaining the temperature at 25°C. Finally, 10 parts earthworm protein were added, the temperature was lowered to 20°C, and the mixture was stirred at a low speed of 10 rpm for 15 minutes. The resulting material was passed through a 20-mesh sieve and compressed into tablets using a rotary tablet press at a pressure of 10 kN, with a tablet weight of 500 mg and a tablet diameter of 12 mm. The finished product is a light yellow to brownish-yellow round tablet with a hardness of 50-80 N, a disintegration time of no more than 30 minutes, and a friability of no more than 0.8%.

[0038] Example 2: Tablets were prepared according to the following formulation. 15 parts earthworm protein, 90 parts total flavonoid extract from sea buckthorn (total flavonoid content 65%), 120 parts silymarin extract (silymarin content 52%), 60 parts maltodextrin, and 45 parts pregelatinized starch were taken. First, 120 parts silymarin extract and 45 parts pregelatinized starch were mixed at room temperature (25°C) for 5 minutes. Then, 90 parts total flavonoid extract from sea buckthorn and 60 parts maltodextrin were added, and the mixture was stirred for 12 minutes. Next, 15 parts earthworm protein were added, the temperature was lowered to 18°C, and the mixture was stirred at low speed for 18 minutes. The material was passed through a 20-mesh sieve and compressed into tablets at a pressure of 12 kN, with a tablet weight of 530 mg and a tablet diameter of 12 mm. The finished product was a brownish-yellow round tablet with a hardness of 55–85 N and a disintegration time not exceeding 25 minutes.

[0039] Example 3: Tablets were prepared according to the following formulation. 8 parts earthworm protein, 50 parts total flavonoid extract from sea buckthorn (total flavonoid content 75%), 80 parts silymarin extract (silymarin content 58%), 35 parts maltodextrin, and 25 parts pregelatinized starch were taken. First, 80 parts silymarin extract and 25 parts pregelatinized starch were mixed at room temperature (25°C) for 4 minutes. Then, 50 parts total flavonoid extract from sea buckthorn and 35 parts maltodextrin were added, and the mixture was stirred for 8 minutes. Next, 8 parts earthworm protein were added, the temperature was lowered to 20°C, and the mixture was stirred at low speed for 12 minutes. The material was passed through a 24-mesh sieve and compressed into tablets at a pressure of 8 kN, with a tablet weight of 400 mg and a tablet diameter of 10 mm. The finished product was a pale yellow, round tablet with a hardness of 45–70 N and a disintegration time not exceeding 35 minutes.

[0040] Example 4: Prepare tablets according to the following formulation. Take 25 parts earthworm protein, 110 parts total flavonoid extract from sea buckthorn (total flavonoid content 62%), 140 parts silymarin extract (silymarin content 50%), 70 parts maltodextrin, and 55 parts pregelatinized starch. First, mix 140 parts silymarin extract and 55 parts pregelatinized starch at room temperature (25°C) for 6 minutes. Add 110 parts total flavonoid extract from sea buckthorn and 70 parts maltodextrin, and stir for 13 minutes. Add 25 parts earthworm protein, cool to 20°C, and stir at low speed for 18 minutes. After passing the material through a 16-mesh sieve, compress it into tablets at a pressure of 14 kN, with a tablet weight of 600 mg and a tablet diameter of 13 mm. The finished product is a brown, round tablet with a hardness of 60-90 N and a disintegration time not exceeding 25 minutes.

[0041] Example 5: Prepare tablets according to the following formulation. Take 20 parts of earthworm protein, 100 parts of total flavonoid extract from sea buckthorn (total flavonoid content 68%), 130 parts of silymarin extract (silymarin content 51%), 65 parts of maltodextrin, and 50 parts of pregelatinized starch. Mix 130 parts of silymarin extract and 50 parts of pregelatinized starch at room temperature (25°C) for 5 minutes. Add 100 parts of total flavonoid extract from sea buckthorn and 65 parts of maltodextrin and stir for 12 minutes. Add 20 parts of earthworm protein, cool to 19°C, and stir at low speed for 16 minutes. After passing the material through an 18-mesh sieve, compress it into tablets at a pressure of 11 kN, with a tablet weight of 550 mg and a tablet diameter of 12 mm. The finished product is a deep yellow round tablet with a hardness of 55-80 N and a disintegration time not exceeding 28 minutes.

[0042] The products in Examples 1 to 5 above are packaged in aluminum-plastic blister packs, with 12 pieces per blister pack and 3 blister packs per box, totaling 36 pieces. The aluminum-plastic blister pack consists of a 0.25mm thick polyvinyl chloride (PVC) base film and 0.02mm thick aluminum foil. The heat-sealing temperature is 150–170℃, and the heat-sealing time is 0.5–1.0s. After packaging, store in a dark, sealed, cool, and dry place, with a storage temperature not exceeding 25℃ and a relative humidity not exceeding 65%. The choice of aluminum-plastic blister packs is based on the following considerations: the active proteins in earthworm protein are sensitive to humidity and oxygen; the high barrier properties of aluminum foil can effectively prevent water vapor and oxygen penetration, delaying protein oxidative denaturation; total flavonoids from sea buckthorn are prone to photochemical degradation under light conditions, and the light-shielding properties of aluminum foil can effectively protect the stability of flavonoid compounds. Compared to bottled packaging, aluminum-plastic blister packs also have advantages such as precise single-piece dosage, convenient carrying, and avoiding the risk of moisture absorption caused by repeated opening.

[0043] A systematic analysis of the preparation process parameters in Examples 1 to 5 revealed that stirring temperature is a key factor affecting the retention of earthworm protein activity. When the stirring temperature in step three exceeds 25°C, the fibrinolytic activity retention rate of earthworm protein drops below 85%; when the temperature is controlled within the range of 18–22°C, the fibrinolytic activity retention rate can reach over 95%. This is because fibrinolytic system-activating proteins are heat-sensitive proteins, and their tertiary structure is prone to irreversible denaturation at high temperatures. Regarding stirring speed, low-speed stirring (5–15 r / min) in step three is used to avoid the shear force generated by high-speed stirring from damaging the protein's spatial conformation. The selection of sieve mesh size (16–24 mesh) ensures both the uniformity of material particle size and avoids frictional heat generation caused by excessively fine sieves. The suitable range for tableting pressure is 5–15 kN; too low a pressure results in loose and brittle tablets, while too high a pressure may compress protein particles, causing activity loss. The comparison of Examples 1 to 5 shows that, within the preferred process parameter range, the product quality is stable and controllable, and the relative standard deviation of fibrinolytic activity between batches does not exceed 8%.

[0044] Comparative Example 1: Using only earthworm protein as a single component, 10 parts earthworm protein, 140 parts maltodextrin, and 120 parts pregelatinized starch were used to prepare tablets according to the preparation method of Example 1. Each tablet weighed 500 mg and did not contain total flavonoids from sea buckthorn or silymarin.

[0045] Comparative Example 2: A two-component combination of earthworm protein and nattokinase was used. 10 parts earthworm protein, 15 parts nattokinase, 125 parts maltodextrin, and 120 parts pregelatinized starch were used to prepare tablets according to the preparation method of Example 1. Each tablet weighed 500 mg and did not contain total flavonoids from sea buckthorn or silymarin.

[0046] Comparative Example 3: A two-component combination of earthworm protein and total flavonoids from sea buckthorn (excluding silymarin) was used. 10 parts earthworm protein, 75 parts total flavonoid extract from sea buckthorn, 90 parts maltodextrin, and 95 parts pregelatinized starch were used to prepare tablets according to the preparation method of Example 1. Each tablet weighed 500 mg.

[0047] Quantitative assessment of synergistic effect: Fibrinolytic activity (dissolution zone area) and thrombus weight reduction rate were used as evaluation indicators. The degree of synergy among components was assessed by calculating the synergy index (CI value) using the Chou-Talalay method. The fibrinolytic activity of Example 1 (complete three-component formulation) was used as the observed value (285 mm). 2 Comparative Example 1 (earthworm protein alone, 165 mm) 2 Comparative Example 3 (earthworm protein + total flavonoids from sea buckthorn, 235mm) 2 The fibrinolytic activity of the three components is a reference value for both single-component and two-component systems. Theoretically, if the three components only exhibit an additive effect, the expected fibrinolytic activity should equal the sum of the independent contributions of each component. However, the measured value is 285 mm. 2 Significantly higher than the theoretical sum of 215 mm 2 (P<0.01), with a co-existence index (CI) of 0.72<1, quantitatively confirming a synergistic effect among the three components rather than a simple additive effect. Regarding the reduction rate of thrombus weight, Example 1 showed a reduction rate of 66.7%, while Comparative Examples 1 to 3 showed reduction rates of 31.2%, 43.5%, and 52.2%, respectively. The theoretically calculated reduction rate based on the independent contributions of the three components was 55.8%. The measured reduction rate of Example 1 exceeded the theoretical value by 10.9 percentage points, further confirming the existence of a synergistic effect. The biological basis of this synergistic effect lies in the fact that silymarin reduces blood viscosity by improving hepatic lipid metabolism, thereby increasing the transport efficiency of earthworm protein and total flavonoids from sea buckthorn to the thrombus site, forming a positive feedback loop.

[0048] Experiment 1: In vitro fibrinolytic activity assay. The fibrinolytic activity of each group of samples was evaluated using the fibrin plate lysis zone method. A fibrinogen solution (3 mg / mL) was mixed with thrombin (10 U / mL) and spread onto a plate to form a fibrin gel layer. Samples from each example and comparative example were ground and prepared into a 10 mg / mL aqueous solution. 20 μL of each solution was spotted onto a fibrin plate, incubated at 37°C for 18 h, and then the lysis zone area was measured. The lysis zone area of ​​Example 1 was 285 ± 18 mm². 2 Example 2 is 312±22mm 2 Example 3 is 248±15mm 2 Example 4 is 335±25mm 2 The melting zone area of ​​Comparative Example 1 (earthworm protein alone) was 165 ± 12 mm. 2 Comparative Example 2 (earthworm protein + nattokinase) was 220±16 mm. 2Comparative Example 3 (earthworm protein + total flavonoids from sea buckthorn) had a content of 235 ± 19 mm. 2 The above data show that the fibrinolytic activity of the three-component synergistic formulation of this invention is significantly better than that of single-component and two-component combinations. Example 1 shows a 72.7% increase compared to Comparative Example 1, a 29.5% increase compared to Comparative Example 2, and a 21.3% increase compared to Comparative Example 3. Figure 1 As shown, the dissolution area of ​​the three-component synergistic formulation exhibits a significant dose-component dependent increasing trend.

[0049] Experiment 2: In vivo thrombolysis experiment in rats. Sixty SD rats weighing 250±20g were randomly divided into 6 groups of 10 each. The blank control group was administered saline by gavage, while the model control group was administered saline by gavage after establishing a carotid artery thrombosis model. Example 1 group was administered the sample from Example 1 (100mg / kg / d), Comparative Example 1 group was administered the sample from Comparative Example 1 (100mg / kg / d), Comparative Example 2 group was administered the sample from Comparative Example 2 (100mg / kg / d), and Comparative Example 3 group was administered the sample from Comparative Example 3 (100mg / kg / d). The thrombosis model was prepared using the FeCl3 injury method: filter paper strips soaked in 10% ferric chloride solution were applied to the outer wall of the left carotid artery of the rats for 10 minutes to induce endothelial damage and thrombus formation. Rats in each group were administered the saline solution for 28 consecutive days before being sacrificed for tissue collection.

[0050] The results of thrombus weight measurement are as follows. The wet weight of the thrombus in the model control group was 18.6±2.3 mg, in Example 1 group it was 6.2±1.4 mg (a decrease of 66.7% compared to the model group, P<0.01), in Comparative Example 1 it was 12.8±1.9 mg (a decrease of 31.2%, P<0.05), in Comparative Example 2 it was 10.5±1.7 mg (a decrease of 43.5%, P<0.01), and in Comparative Example 3 it was 8.9±1.5 mg (a decrease of 52.2%, P<0.01). The differences between Example 1 group and each comparative example group were statistically significant (P<0.05), confirming that the in vivo thrombolytic effect of the three-component synergistic formulation was significantly better than that of single-component and two-component combinations. Figure 2 As shown, the wet weight of the thrombus in Example 1 group was the lowest among all experimental groups, which is in stark contrast to the comparative groups.

[0051] Histopathological observation: After the rats in each group were sacrificed, the thrombus segment of the left common carotid artery was embedded in paraffin, sectioned (5 μm thick), and stained with hematoxylin and eosin (HE). Figure 6A In the model control group, large areas of red thrombus adhesion were observed in the blood vessel lumen. The thrombus was rich in red blood cells and fibrin mesh structure. Endothelial cells on the surface of the blood vessel intima were severely detached, and the internal elastic membrane was wrinkled. Figure 6DIn Example 1, the residual thrombus area in the vascular lumen was reduced by about 70% compared with the model group. The thrombus structure was loose, the fibrin mesh was significantly degraded and broken, and new endothelial cells were visible covering the vascular intima surface. The morphology of the internal elastic membrane was close to normal. Figure 6B The thrombus area in the vascular lumen of the control group was reduced by about 30% compared with that of the model group, and the thrombus structure was still relatively dense. Figure 6C In Comparative Example 3, the thrombus area within the vascular lumen was reduced by approximately 50%, and the degree of endothelial repair was between that of Example 1 and Comparative Example 1. The above pathological observations directly confirmed the advantages of the three-group formulation of this invention in promoting thrombus degradation and vascular endothelial repair at the tissue morphology level. Particularly noteworthy is that the degree of vascular endothelial repair in Example 1 was significantly better than that in Comparative Example 1 and Comparative Example 3, which is closely related to the synergistic pharmacological effects of silymarin and total flavonoids from sea buckthorn in reducing oxidative stress and alleviating endothelial cell inflammation. In Example 1, the vascular lumen was patent and the endothelium was continuous and intact, forming a stark contrast to the pathological manifestations of large-area thrombus adhesion and severe endothelial detachment in the model control group.

[0052] Experiment 3: Blood Lipid Level Detection. After 28 days of gavage administration to rats in each group of the above experiments, abdominal aortic blood was collected, and plasma was separated to detect four blood lipid indicators. In Example 1 group, the plasma total cholesterol (TC) was 2.15±0.28 mmol / L, triglycerides (TG) was 0.68±0.12 mmol / L, low-density lipoprotein cholesterol (LDL-C) was 0.85±0.14 mmol / L, and high-density lipoprotein cholesterol (HDL-C) was 1.62±0.18 mmol / L. In the model control group, the TC was 4.85±0.56 mmol / L, TG was 1.52±0.23 mmol / L, LDL-C was 2.78±0.32 mmol / L, and HDL-C was 0.95±0.12 mmol / L. In Comparative Example 1, the total cholesterol (TC) was 4.12 ± 0.48 mmol / L, total triglycerides (TG) was 1.35 ± 0.19 mmol / L, LDL-C was 2.45 ± 0.28 mmol / L, and HDL-C was 1.08 ± 0.14 mmol / L. In Comparative Example 2, the TC was 3.86 ± 0.42 mmol / L, TG was 1.28 ± 0.17 mmol / L, LDL-C was 2.32 ± 0.26 mmol / L, and HDL-C was 1.12 ± 0.15 mmol / L. In Comparative Example 3, the TC was 2.95 ± 0.35 mmol / L, TG was 0.92 ± 0.15 mmol / L, LDL-C was 1.55 ± 0.20 mmol / L, and HDL-C was 1.38 ± 0.16 mmol / L.

[0053] Data showed that Example 1 group was significantly superior to the comparative groups in reducing TC, TG, and LDL-C, and increasing HDL-C (P<0.05), confirming that the addition of silymarin produced a synergistic effect superimposed on the lipid-lowering effect of total flavonoids from sea buckthorn by enhancing hepatic lipid metabolism. The LDL-C level in Example 1 group was reduced by 69.4% compared to the model control group, while the reduction in Comparative Group 3 (without silymarin) was only 44.2%. The difference in LDL-C levels between the two groups was statistically significant (P<0.01), indicating that the contribution of silymarin to the upregulation of hepatic LDL receptors is irreplaceable. Figure 3 As shown, the levels of TC, TG, and LDL-C in Example 1 group were at their lowest values, while the level of HDL-C was at its highest value, exhibiting a fully optimized lipid profile.

[0054] Experiment 4: Detection of t-PA and PAI-1 activities. The activities of t-PA and PAI-1 in the plasma of rats in each group were determined using a chromogenic substrate method. In Example 1 group, the plasma t-PA activity was 0.85±0.09 IU / mL, and the PAI-1 activity was 12.3±1.8 AU / mL. In the model control group, the t-PA activity was 0.32±0.05 IU / mL, and the PAI-1 activity was 28.6±3.5 AU / mL. In Comparative Example 1 group, the t-PA activity was 0.58±0.07 IU / mL, and the PAI-1 activity was 20.2±2.6 AU / mL. In Comparative Example 2 group, the t-PA activity was 0.62±0.08 IU / mL, and the PAI-1 activity was 18.8±2.4 AU / mL. In Comparative Example 3, the t-PA activity was 0.72 ± 0.08 IU / mL, and the PAI-1 activity was 16.5 ± 2.1 AU / mL. In Example 1, the t-PA activity was 165.6% higher than the model control group, 46.6% higher than Comparative Example 1, and 18.1% higher than Comparative Example 3. Regarding PAI-1 activity, Example 1 showed a 57.0% decrease compared to the model control group and a 39.1% decrease compared to Comparative Example 1. The t-PA / PAI-1 ratio was 0.069 in Example 1, 0.011 in the model control group, 0.029 in Comparative Example 1, and 0.044 in Comparative Example 3. The three-component formulation of this invention increased the t-PA / PAI-1 ratio to 6.3 times that of the model group, significantly better than the 2.6 times of Comparative Example 1 and the 4.0 times of Comparative Example 3, indicating that the synergistic effect of the three components comprehensively activated the endogenous fibrinolytic system. Figure 4 As shown, the t-PA activity bar chart and the PAI-1 activity bar chart show opposite trends, and the t-PA / PAI-1 ratio of the Example 1 group is the highest among all groups.

[0055] Experiment 5: Detection of the PGI2 / TXA2 ratio. The levels of 6-keto-prostaglandin F1α (a stable metabolite of PGI2) and thromboxane B2 (a stable metabolite of TXA2) in the plasma of rats in each group were detected using enzyme-linked immunosorbent assay (ELISA), and the PGI2 / TXA2 ratio was calculated. In Example 1 group, the PGI2 metabolite level was 358.5±42.6 pg / mL, and the TXA2 metabolite level was 142.8±18.5 pg / mL, with a ratio of 2.51. In the model control group, the PGI2 metabolite level was 185.2±25.3 pg / mL, and the TXA2 metabolite level was 315.6±38.2 pg / mL, with a ratio of 0.59. In Comparative Example 1 group, the PGI2 metabolite level was 198.3±28.5 pg / mL, and the TXA2 metabolite level was 288.5±32.4 pg / mL, with a ratio of 0.69. In Comparative Example 3, the PGI2 metabolite level was 305.8 ± 36.8 pg / mL, and the TXA2 metabolite level was 168.2 ± 22.1 pg / mL, with a ratio of 1.82. In Example 1, the PGI2 / TXA2 ratio was 2.51, an increase of 325.4% compared to the model group and 37.9% compared to Comparative Example 3 (without silymarin). These results confirm that total flavonoids from sea buckthorn effectively regulated prostaglandin balance in the formulation, while silymarin further optimized the vascular endothelial environment by improving hepatic lipid metabolism, indirectly contributing to the increase in the PGI2 / TXA2 ratio. Figure 5 As shown, the PGI2 / TXA2 ratio of the Example 1 group was much higher than that of the model control group and each comparative group, which directly reflects the synergistic effect of total flavonoids from sea buckthorn and silymarin in regulating prostaglandin metabolism balance.

[0056] Supplement to Experiment 5: Dose-response relationship investigation. Based on the formulation in Example 1, in vivo thrombolysis experiments were conducted in rats with low-dose groups (50 mg / kg / d), medium-dose groups (100 mg / kg / d), and high-dose groups (200 mg / kg / d). The wet weight of the thrombus in the low-dose group was 11.5 ± 1.8 mg (a decrease of 38.2% compared to the model group), in the medium-dose group it was 6.2 ± 1.4 mg (a decrease of 66.7%), and in the high-dose group it was 4.8 ± 1.1 mg (a decrease of 74.2%). Regarding t-PA activity, the low-dose group was 0.55 ± 0.06 IU / mL, the medium-dose group was 0.85 ± 0.09 IU / mL, and the high-dose group was 0.92 ± 0.10 IU / mL. Regarding TC levels, the low-dose group was 3.25±0.38 mmol / L, the medium-dose group was 2.15±0.28 mmol / L, and the high-dose group was 1.95±0.25 mmol / L. These data indicate that the thrombolytic and lipid-lowering effects of the formulation of this invention exhibit a dose-dependent characteristic. The improvement in effect between the medium-dose and high-dose groups tends to level off, indicating that 100 mg / kg / day is the optimal dose range for efficiency. Based on the human equivalent dose conversion factor (rat coefficient 6.2), the recommended daily human dosage is 2–3 tablets (500 mg per tablet), which corresponds to a daily intake of 20–30 mg of earthworm protein, 150–225 mg of total flavonoids from sea buckthorn, and 200–300 mg of silymarin.

[0057] Experiment 6: Safety Evaluation. Following the requirements of the acute toxicity test, 40 ICR mice (half male and half female) were administered the suspension of the sample from Example 1 at the maximum gavage volume (40 mL / kg) three times a day, with a total dose of 20 g / kg. General behavior, weight changes, and mortality were observed over 14 days. Results showed that all mice survived, gained weight normally, and showed no abnormal behavior, poisoning symptoms, or death. Gross anatomical examination of any organs revealed no visible abnormalities. Following the requirements of the 30-day feeding test, 80 SD rats were divided into four groups (control group, low-dose group 1.67 g / kg / day, medium-dose group 3.33 g / kg / day, and high-dose group 10.0 g / kg / day), and administered the suspension by gavage for 30 consecutive days. After the experiment, the complete blood count and blood biochemical indicators (including alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (CRE), and blood urea nitrogen (BUN) of all groups were within the normal range, with no statistically significant differences between groups (P>0.05). No pathological changes related to the test sample were found in the histopathological examination of various organs, confirming that the formulation of the present invention has good safety at both the recommended and high doses.

[0058] Supplementary safety evaluation: Genotoxicity test. The mutagenicity of the samples from Example 1 was detected using the Ames test (Salmonella Typhimurium strains TA97, TA98, TA100, and TA102, with and without the S9 metabolic activation system). Within the dosage range of 0.05–5.0 mg / plate, the number of revertant colonies for each strain did not exceed twice the number of spontaneously revertant colonies, and there was no dose-response relationship; the Ames test results were negative. In the mouse bone marrow micronucleus test, ICR mice were orally administered three doses (2.5 g / kg, 5.0 g / kg, and 10.0 g / kg) for 2 consecutive days. Six hours after the last administration, the mice were sacrificed, and bone marrow smears were collected. The micronucleus rate in each dose group (1.2‰–1.8‰) was not statistically different from the negative control group (1.0‰) (P>0.05), indicating that the formulation of this invention has no genotoxicity. In the mouse sperm abnormality test, after continuous gavage administration of the same dose for 35 days, there was no statistically significant difference in sperm abnormality rate between the dose groups (1.8%–2.2%) and the negative control group (1.6%) (P>0.05), further confirming the safety of the formulation of the present invention.

[0059] Coagulation function safety assessment: Considering the dual effects of the formulation of this invention in promoting fibrinolysis and inhibiting platelet aggregation, a systematic evaluation of coagulation function indicators was conducted. Forty SD rats were randomly divided into a control group and a high-dose group (200 mg / kg / d) as described in Example 1, and administered the drugs by gavage for 60 consecutive days. After the experiment, four coagulation indicators were measured: prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB) levels. The results showed that in the high-dose group, PT was 12.8 ± 0.9 s (control group 12.5 ± 0.8 s), APTT was 28.5 ± 2.3 s (control group 27.8 ± 2.1 s), TT was 18.2 ± 1.5 s (control group 17.9 ± 1.4 s), and FIB was 2.85 ± 0.32 g / L (control group 2.92 ± 0.28 g / L). All indicators were within the normal range, and there was no statistically significant difference between the two groups (P>0.05). The above results confirm that the formulation of the present invention can effectively activate the fibrinolytic system without causing abnormal coagulation function or increased bleeding risk. Its thrombolytic mechanism is achieved by gently enhancing endogenous fibrinolytic capacity rather than directly interfering with the coagulation cascade reaction, thus having a significant safety advantage over chemical thrombolytic drugs.

[0060] Experiment 7: Stability Assessment. The product from Example 1 was placed in aluminum-plastic blister packaging and stored for 6 months under accelerated conditions (temperature 40±2℃, relative humidity 75±5%) and 12 months under long-term conditions (temperature 25±2℃, relative humidity 60±5%). Samples were taken every 3 months to test appearance, disintegration time, moisture content, total flavonoid content, and fibrinolytic activity of earthworm protein. After 6 months of accelerated testing, there was no significant change in appearance. The disintegration time increased from 28 min to 31 min (still meeting the requirement of not exceeding 60 min), the moisture content increased from 3.2% to 4.1% (still below the 5% limit), the total flavonoid content decreased from 42.5 mg / tablet to 39.8 mg / tablet (retention rate 93.6%), and the fibrinolytic activity decreased from a disintegration zone area of ​​282 mm². 2 Reduced to 258mm 2 (Retention rate 91.5%). After a long-term test of 12 months, the changes in all indicators were within acceptable ranges, with a total flavonoid retention rate of 96.2% and a fibrinolytic activity retention rate of 94.8%, confirming that the formulation of this invention has a shelf life of not less than 24 months under the recommended packaging conditions.

[0061] In vitro disintegration and dissolution testing: Following the disintegration time test method of the 2020 edition of the Chinese Pharmacopoeia, six tablets from each of Examples 1 to 5 were tested. The disintegration time for Example 1 was 28±3 min, for Example 2 it was 24±2 min, for Example 3 it was 33±3 min, for Example 4 it was 23±2 min, and for Example 5 it was 26±3 min, all meeting the limit requirement of not exceeding 60 min. Dissolution testing (paddle method, rotation speed 50 r / min, dissolution medium 900 mL pH 6.8 phosphate buffer, temperature 37±0.5℃) was used to detect the dissolution curves of total flavonoids in the tablets of each example. For Example 1, the cumulative dissolution rate of total flavonoids was 35.8% at 15 min, 62.4% at 30 min, 85.6% at 45 min, and 93.2% at 60 min. The cumulative dissolution rates at 60 min for Examples 2 to 5 were 95.1%, 88.5%, 96.8%, and 94.3%, respectively, all meeting the standard of a cumulative dissolution rate of not less than 75% at 45 min. The release curves of the fibrinolytic activity of earthworm protein showed that the fibrinolytic activity release rate was 58.3% at 30 min and 89.7% at 60 min in pH 6.8 buffer, indicating that the disintegration-promoting effect of pregelatinized starch and the dispersing and solubilizing effect of maltodextrin effectively ensured the full release of the active components.

[0062] The quality control methods are as follows. The fibrinolytic activity of earthworm protein was determined using the fibrin plate method, with a standard curve established using urokinase as a standard. The fibrinolytic activity of each tablet composition was not less than 80 IU. The total flavonoid content of sea buckthorn was determined using high-performance liquid chromatography (HPLC), with a C24 column. 18The column was 250 mm × 4.6 mm, 5 μm, the mobile phase was methanol-0.1% phosphoric acid aqueous solution (v / v 55:45), the detection wavelength was 370 nm, and the total flavonoid content was calculated using rutin as a reference standard. The total flavonoid content in each tablet composition was not less than 40 mg. Silymarin content was determined by HPLC using a C10 column. 18 The column (250 mm × 4.6 mm, 5 μm) was used with a mobile phase of methanol-water (48:52 v / v). The detection wavelength was 288 nm. Quantification was performed using the external standard method with silymarin standard. The silymarin content in each tablet composition was not less than 25 mg. Microbiological limits were set according to General Chapters 1105 and 1106 of the 2020 edition of the Chinese Pharmacopoeia: total bacterial count not exceeding 1000 CFU / g, total mold and yeast count not exceeding 100 CFU / g, and Escherichia coli not detectable.

[0063] Moisture content was determined using the drying method (General Chapter 0832, Chinese Pharmacopoeia 2020 Edition), drying at 105℃ to constant weight, with a moisture content not exceeding 5.0%. Tablet weight variation was tested according to General Chapter 0101, Chinese Pharmacopoeia 2020 Edition, precisely weighing 20 tablets each, with a weight variation limit of ±5%. The number of tablets exceeding this limit should not exceed 2 tablets and should not exceed twice the limit. Friability was tested according to General Chapter 0923, Chinese Pharmacopoeia 2020 Edition, placing 10 pre-weighed tablets in a friability tester, running at 25 r / min for 100 cycles, then removing fragments and weighing again; friability should not exceed 1.0%. Heavy metal and harmful element testing was conducted according to General Chapter 2321, Chinese Pharmacopoeia 2020 Edition, with lead content not exceeding 5.0 mg / kg, cadmium content not exceeding 1.0 mg / kg, arsenic content not exceeding 2.0 mg / kg, mercury content not exceeding 0.2 mg / kg, and copper content not exceeding 20.0 mg / kg. Pesticide residue testing was conducted in accordance with General Chapter 2341 of the 2020 edition of the Chinese Pharmacopoeia. Residues of organochlorine, organophosphorus, and pyrethroid pesticides must all meet the limit requirements. This quality control system ensures traceability and batch-to-batch consistency of the product at every stage, from raw materials to finished products.

[0064] Supplementary efficacy verification experiment: Antioxidant index detection. Plasma and liver tissue homogenates were collected from rats in each group, and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) content were measured. Regarding plasma SOD activity, the activity was 185.3±15.6 U / mL in Example 1, 112.5±10.8 U / mL in the model control group, 128.6±12.3 U / mL in Comparative Example 1, and 158.2±14.1 U / mL in Comparative Example 3. Regarding plasma MDA content, the activity was 3.85±0.42 nmol / mL in Example 1, 8.56±0.95 nmol / mL in the model control group, 7.12±0.78 nmol / mL in Comparative Example 1, and 5.28±0.58 nmol / mL in Comparative Example 3. Regarding liver tissue GSH-Px activity, the levels in Example 1 were 268.5±22.4 U / mg protein, the model control group was 145.8±13.6 U / mg protein, Comparative Example 1 was 162.3±15.2 U / mg protein, and Comparative Example 3 was 215.6±18.8 U / mg protein. The data showed that the antioxidant indicators of Example 1 were comprehensively superior to those of the comparative groups, with liver tissue GSH-Px activity increasing by 24.5% compared to Comparative Example 3 (P<0.05). This confirms that silymarin enhances the overall antioxidant defense capacity of the liver by directly protecting the hepatocyte antioxidant enzyme system, complementing the plasma antioxidant effect of total flavonoids from sea buckthorn, and jointly reducing systemic oxidative stress levels. Figure 7 As shown, the SOD activity and GSH-Px activity of the group in Example 1 were the highest among all groups, while the MDA content was the lowest, confirming that the three groups have comprehensive antioxidant protection effects.

[0065] Comprehensive Mechanism Analysis: The formulation of this invention achieves a three-dimensional targeted synergistic effect of "fibrinolysis activation - antithrombosis - metabolic regulation". At the fibrinolysis activation level, the fibrinolytic system activating protein in earthworm protein promotes the release of t-PA from vascular endothelial cells, initiating the plasminogen → plasmin cascade reaction, directly degrading existing fibrin thrombi and restoring vascular patency. At the antithrombosis level, isorhamnetin and quercetin in total flavonoids of sea buckthorn regulate the cyclooxygenase metabolic pathway, increasing the PGI2 / TXA2 ratio and inhibiting excessive platelet aggregation, intervening at the initiation stage of thrombosis. Simultaneously, their effects of reducing TC and TG decrease lipid deposition in the vascular intima, delaying the progression of atherosclerosis. At the metabolic regulation level, silymarin enhances the liver's ability to clear LDL-C by protecting hepatocyte integrity, promoting liver protein synthesis, and upregulating LDL receptor expression, eliminating risk factors for thrombosis from the perspective of systemic lipid metabolism. The three levels of action are independent yet synergistic: earthworm protein dissolves existing thrombi, total flavonoids from sea buckthorn prevent the formation of new thrombi, and silymarin eliminates the metabolic causes that promote thrombus formation, forming a complete "thrombolysis-thrombus prevention-cause control" intervention loop. Experimental data confirm that the thrombolytic effect and lipid-lowering effect of the three-component combination are significantly better than any single-component or two-component combination, demonstrating the non-linear synergistic effect of multi-component, multi-target synergy.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A formula for enhancing the body's own thrombolytic ability, characterized in that, The product comprises, by weight, the following components: 5-30 parts earthworm protein, 30-120 parts total flavonoid extract from sea buckthorn, 50-150 parts silymarin extract, 20-80 parts maltodextrin, and 15-60 parts pregelatinized starch; wherein the earthworm protein contains no less than 15% fibrinolytic system activating protein of the total protein content, the total flavonoid extract from sea buckthorn contains no less than 60% total flavonoid content, and the silymarin extract from sea buckthorn contains no less than 50% silymarin.

2. The formulation according to claim 1, characterized in that, By weight, earthworm protein 8-15 parts, total flavonoid extract of sea buckthorn 50-90 parts, silymarin extract 80-120 parts, maltodextrin 35-60 parts, and pregelatinized starch 25-45 parts.

3. The formulation according to claim 1, characterized in that, The fibrinolysis system activating protein in the earthworm protein has a molecular weight range of 20-35 kDa. The earthworm protein is obtained by low-temperature water extraction, enzymatic hydrolysis, ultrafiltration separation and freeze drying of fresh Eisenia fetida or Panax ginseng. The protein purity is not less than 85%.

4. The formulation according to claim 1, characterized in that, The active ingredients in the total flavonoid extract of sea buckthorn include isorhamnetin, quercetin, myricetin and kaempferol, with the total content of the four aglycone compounds accounting for more than 40% of the total flavonoid content.

5. The formulation according to claim 1, characterized in that, The formulation is in tablet form, with a tablet weight of 400–600 mg.

6. A method for preparing the formulation according to any one of claims 1 to 5, characterized in that, Includes the following steps: Step 1: Mix silymarin extract and pregelatinized starch evenly at 25±5℃ and stir for 3-8 minutes. Step 2: Add total flavonoid extract of sea buckthorn and maltodextrin to the mixture obtained in Step 1 and stir at 25±5℃ for 8-15 minutes. Step 3: Add earthworm protein to the mixture obtained in Step 2 and stir at low speed for 12-20 minutes at 20±3℃. Step 4: Pass the material obtained in Step 3 through a 16-24 mesh sieve, mix evenly, and then directly compress into tablets.

7. The method according to claim 6, characterized in that, In step four, the tableting pressure is 5–15 kN, the hardness of the resulting tablets is 45–90 N, and the disintegration time does not exceed 35 min.

8. The method according to claim 6, characterized in that, In step three, the stirring speed is 5-15 r / min, and the earthworm protein is pre-cooled at 4℃ for no less than 30 min before being added.

9. The method according to claim 6, characterized in that, In step four, the material is directly compressed into tablets, eliminating the need for a granulation step.

10. The application of the formulation according to any one of claims 1 to 5 in the preparation of functional foods or health foods that enhance the activity of the human fibrinolytic system.

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