A composition containing earthworm protein with thrombosis-reducing function and its preparation method and application

Abstract

The application discloses a composition containing earthworm protein and having a function of reducing thrombus, and a preparation method and application thereof. The composition is prepared from mulberry leaves, Chinese sumac leaves, casein hydrolysate, earthworm protein, perilla and the like in specific proportions. The application adopts a segmented preparation process. The raw materials such as the mulberry leaves and the Chinese sumac leaves are subjected to synergistic fermentation of Aspergillus oryzae and Bacillus velezensis, the perilla and the rosehip are subjected to low-temperature ultrasonic ethanol extraction to retain active ingredients, and finally, the natto kinase is compounded to obtain the composition product. The components are synergistically enhanced, can significantly prolong the blood clotting time, promote fibrinolysis, have excellent thrombus-reducing activity, and can be used for preparing medicines or health products for preventing and improving thrombus-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of functional composition technology, specifically relating to a composition containing earthworm protein with antithrombotic function, its preparation method and application. Background Technology

[0002] Thrombotic diseases are prevalent and endanger human cardiovascular health. They are mainly caused by abnormal platelet aggregation, fibrin metabolism disorders, and imbalance of the vascular microenvironment. They are characterized by high incidence, high disability rate, and high recurrence rate, seriously threatening human life and health. Currently, most clinical antithrombotic drugs are chemical drugs, which have drawbacks such as bleeding risk, heavy metabolic burden on the liver and kidneys, and unsuitability for long-term daily management.

[0003] Food and medicinal ingredients have become a research hotspot for antithrombotic functional compositions due to their high safety, long-term suitability, and multi-target regulation. However, existing antithrombotic compound ingredient formulations have several shortcomings: most formulations have arbitrary ingredient ratios, failing to form a synergistic system; their processing techniques are often simplistic, relying on direct pulverization or simple water extraction, which fails to fully release the active ingredients such as flavonoids and polyphenols from plant materials and is also difficult to degrade large animal protein molecules, resulting in low activity utilization; some formulations suffer from severe homogenization of processing steps, leading to weak antithrombotic effects and poor stability.

[0004] Meanwhile, existing technologies rarely employ refined, segmented processing techniques for compound systems containing mulberry leaves, Eucommia ulmoides leaves, earthworm protein, and nattokinase. This hinders the simultaneous extraction of active substances from plant materials, low-temperature preservation of active ingredients, and deep activation of proteins and peptides, preventing the full realization of synergistic effects among components and significantly limiting the antithrombotic efficacy of composite functional compositions. Currently, most antithrombotic compound preparations rely on a single anticoagulant or thrombolytic mechanism for efficacy, failing to synergistically regulate vascular damage and fibrin deposition pathways associated with thrombosis. Therefore, this invention develops a scientifically formulated, optimized, and standardized microbial fermentation system for antithrombotic composition containing earthworm protein, along with its preparation method. This composition exhibits multi-target efficacy and significant synergistic effects. Summary of the Invention

[0005] To solve the aforementioned problems, the present invention adopts the following technical solution: This invention provides a composition with antithrombotic function, which is prepared from the following raw materials in parts by weight: 8-16 parts of mulberry leaves, 6-10 parts of Eucommia ulmoides leaves, 2-6 parts of casein hydrolysate peptides, 4-6 parts of earthworm protein, 8-12 parts of perilla, 5-9 parts of prickly pear, and 0.5-2 parts of nattokinase.

[0006] Furthermore, the composition is prepared from the following raw materials in parts by weight: 12 parts mulberry leaves, 8 parts Eucommia ulmoides leaves, 4 parts casein hydrolysate peptides, 5 parts earthworm protein, 10 parts perilla, 7 parts prickly pear, and 1 part nattokinase.

[0007] Furthermore, the composition also contains pharmaceutically acceptable excipients.

[0008] Furthermore, the composition can be prepared into any one of the following dosage forms: powder, granules, tablets, capsules, or oral liquid.

[0009] The present invention also provides a method for preparing the composition with the function of reducing thrombosis, characterized by comprising the following steps: (1) pretreatment of raw materials; (2) preparation of extract powder a by microbial fermentation; (3) preparation of extract powder b by low temperature ultrasonic synergistic extraction; (4) homogenization of extract powder a, extract powder b and nattokinase to obtain the composition.

[0010] Further, the raw material pretreatment in step (1) includes: weighing each part by weight of the clean dry raw material of the composition, crushing and sieving it and storing it for later use.

[0011] Further, step (2) microbial fermentation includes: mixing pretreated mulberry leaves, Eucommia ulmoides leaves, casein hydrolysate peptides, and earthworm protein, and preparing a suspension with purified water at a material-to-liquid ratio of 1:6-10.

[0012] Further, the activated Aspergillus oryzae ATCC56747 and the activated Bacillus belyssus CICC21430 bacterial solution were mixed at a volume ratio of 3:1 to obtain a composite bacterial solution, which was then added to the suspension for fermentation at an inoculum volume of 4-8% v / v.

[0013] Furthermore, the fermentation was carried out at a constant temperature of 30°C, pH 6.5, and 180 r / min for 72 h.

[0014] Furthermore, after fermentation, the fermentation broth was inactivated by incubation at 90℃ for 15 min, cooled, centrifuged at 8000 r / min for 20 min, and then filtered through a 0.22 μm ceramic membrane. The filtrate was then freeze-dried under vacuum to obtain extract powder a.

[0015] Furthermore, the concentration of the activated Bacillus belyceca CICC21430 bacterial solution is 1×10⁻⁶. 8 The concentration of the activated Aspergillus oryzae ATCC56747 spore suspension was 1×10 CFU / mL. 7 Spores / mL.

[0016] Further, step (3) low-temperature ultrasonic synergistic extraction includes: adding the pretreated perilla and prickly pear powder to a 45% ethanol-purified aqueous solution at a material-liquid ratio of 1:6-10, and extracting for 40 min at 40℃, ultrasonic power of 250W, and ultrasonic frequency of 40kHz.

[0017] Further, the extract was collected by filtration, the residue was extracted once more, and the extracts were combined.

[0018] Further, the combined extracts were centrifuged at 8000 r / min for 20 min, filtered through a 0.22 μm ceramic membrane, and then freeze-dried under vacuum to obtain extract powder b.

[0019] The present invention also provides the use of the composition in the preparation of drugs or health products with antithrombotic function.

[0020] Furthermore, the thrombosis-reducing function includes inhibiting platelet aggregation and promoting fibrinolysis.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention achieves precise synergistic effects among its components by limiting the proportions of each raw material, employing segmented processing techniques, and using a specific compound microbial fermentation system. Mulberry leaves and Eucommia ulmoides leaves provide flavonoid polyphenols for antioxidant and anticoagulant activity; earthworm protein and casein hydrolysate provide small-molecule thrombolytic peptides; and perilla and prickly pear are rich in active substances that improve the vascular microenvironment. These multiple components complement each other and adapt to the multi-pathway regulatory mechanism of thrombosis. Cellular and in vivo experiments have shown that the composition of this invention can significantly synergistically prolong clotting time and increase fibrinolysis rate, demonstrating high application value and promising prospects in the field of thrombosis prevention and treatment.

[0022] (2) The compound of this invention adopts a graded preparation process, and the raw materials are treated separately according to their physicochemical properties: earthworm protein, casein hydrolysate peptide, mulberry leaf, and Eucommia ulmoides leaf are bio-fermented with a compound strain of Aspergillus oryzae and Bacillus belye to enhance the absorption and utilization rate of macromolecular peptides and reduce the irritation of raw materials; perilla and prickly pear are preserved by low-temperature closed ultrasonic extraction throughout the process, and the ethanol-water mixture system can simultaneously dissolve the water-soluble and weakly fat-soluble active substances of the raw materials; nattokinase is a highly active heat-sensitive enzyme preparation, which is not fermented or heated throughout the process, and is only mixed and compounded at the final low temperature. The overall preparation process of this invention avoids the defects of heat-sensitive component inactivation, low utilization rate of active ingredients, and single component action. The process is rigorous, the parameters are quantified, and the repeatability is strong, making it suitable for large-scale mass production. Example

[0023] The present invention will be further illustrated below with reference to specific embodiments. Unless otherwise specified, the raw materials, reagents or devices used in the following embodiments can be obtained from conventional commercial sources or by existing known methods.

[0024] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0025] The microbial strains used in this invention were all purchased from Shanghai Shifeng Biotechnology Co., Ltd., including *Aspergillus oryzae* ATCC56747, *Aspergillus niger* ATCC16404, *Bacillus belyssus* CICC21430, and *Bacillus subtilis* ATCC6633. Microbial activation method: Each strain was inoculated into its corresponding culture medium for activation and adjustment to the target concentration. The concentration of *Bacillus belyssus* and *Bacillus subtilis* was 1×10⁻⁶. 8 CFU / mL, the concentration of Aspergillus oryzae and Aspergillus niger spore suspensions was 1×10⁻⁶. 7 Spores / mL, store for later use.

[0026] The raw materials for the composition of this invention are sourced as follows: mulberry leaves, Eucommia ulmoides leaves, prickly pear and perilla leaves were purchased from Guangzhou Qingping Chinese Herbal Medicine Market; casein hydrolysate peptides were purchased from Gansu Huaan Biotechnology Group; earthworm protein was purchased from Shaanxi Sinote Biotechnology Co., Ltd.; and nattokinase was purchased from Jiangsu Aofu Biotechnology Co., Ltd., with an enzyme activity ≥20000FU / g.

[0027] The kits and reagents of this invention are: Activated Partial Thromboplastin Time (APTT) Assay Kit (Catalog No.: YZ-APTT-001, Shanghai Yuanye Biotechnology Co., Ltd.); Prothrombin Time (PT) Assay Kit (Catalog No.: YZ-PT-001, Shanghai Yuanye Biotechnology Co., Ltd.); Fibrinolysis Rate Assay Kit (Catalog No.: FIB-008, Nanjing Jiancheng Bioengineering Institute); Aspirin (National Drug Approval Number H11021492, Bayer Healthcare Co., Ltd.).

[0028] Example 1 The raw materials in the composition of this embodiment are as follows by weight: 12 parts mulberry leaves, 8 parts Eucommia ulmoides leaves, 4 parts casein hydrolysate peptides, 5 parts earthworm protein, 10 parts perilla, 7 parts prickly pear, and 1 part nattokinase.

[0029] The preparation method of the antithrombotic composition in this embodiment includes the following steps: (1) Raw material pretreatment: Take clean dried raw materials of each weight, crush mulberry leaves, Eucommia ulmoides leaves, perilla and prickly pear into powder and pass through a 40-mesh standard sieve, and pass earthworm protein, casein hydrolysate and nattokinase through a 60-mesh sieve. Store in a sealed container at low temperature and away from light for later use.

[0030] (2) Mixed fermentation of mulberry leaves, Eucommia ulmoides leaves, casein hydrolysate peptides and earthworm protein: After mixing the pretreated mulberry leaves, Eucommia ulmoides leaf powder, casein hydrolysate peptides and earthworm protein, they were mixed with purified water at a ratio of 1:8 to form a suspension. The activated bacterial solutions of Aspergillus oryzae ATCC56747 and Bacillus vesiculosus CICC21430 were taken and mixed at a volume ratio of 3:1 to obtain a compound bacterial solution. The compound bacterial solution was added to the suspension at an inoculum of 6% (v / v) and fermented at a constant temperature of 30℃, pH 6.5 and 180r / min for 72h. After fermentation, the bacterial strain was completely inactivated by keeping the temperature at 90℃ for 15min. After cooling, the mixture was centrifuged at 8000r / min for 20min. The supernatant was filtered through a 0.22μm ceramic membrane and the filtrate was freeze-dried under vacuum to obtain extract powder a.

[0031] (3) Low-temperature ultrasonic synergistic extraction of perilla and prickly pear: The pretreated perilla and prickly pear powders were put into a closed ultrasonic extraction tank, and 45% ethanol-purified water solution (ethanol:purified water = 45:55) was added at a material-liquid ratio of 1:10. Ultrasonic assisted extraction was started at 40℃ with ultrasonic power of 250W and ultrasonic frequency of 40kHz. The extraction was carried out for 40 min each time. The extract was collected by filtration, and the filter residue was extracted again under the same conditions as above. The two extracts were combined. The extract was centrifuged at 8000r / min for 20 min. The supernatant was taken and filtered through a 0.22μm ceramic membrane. The filtrate was freeze-dried under vacuum to obtain extract powder b.

[0032] (4) Mix the composition: Take extract powder a and extract powder b, add 1 part of nattokinase, mix evenly, pass through an 80-mesh sieve to obtain composition S1 with antithrombotic function, and freeze for later use.

[0033] Example 2 The raw materials in this embodiment are as follows (by weight): 16 parts mulberry leaves, 10 parts Eucommia ulmoides leaves, 6 parts casein hydrolysate peptides, 6 parts earthworm protein, 12 parts perilla, 9 parts prickly pear, and 2 parts nattokinase. The preparation method of the antithrombotic composition in this embodiment is the same as in Example 1, resulting in composition S2 with antithrombotic function.

[0034] Example The raw materials of the composition in this embodiment are as follows (by weight): 8 parts mulberry leaves, 6 parts Eucommia ulmoides leaves, 2 parts casein hydrolysate peptides, 4 parts earthworm protein, 8 parts perilla leaves, 5 parts prickly pear, and 0.5 parts nattokinase. The preparation method of the antithrombotic composition in this embodiment is the same as in Example 1, resulting in composition S3 with antithrombotic function.

[0035] Comparative Example 1 The raw materials in this comparative composition by weight are: 12 parts mulberry leaves, 8 parts Eucommia ulmoides leaves, 4 parts casein hydrolysate peptides, 5 parts earthworm protein, 10 parts perilla, 7 parts prickly pear, and 1 part nattokinase.

[0036] The difference between this comparative example and Example 1 is that: in step (2), a mixed bacterial solution of Aspergillus niger ATCC16404 and Bacillus vesiculosus CICC21430 with a volume ratio of 3:1 was used for fermentation. The rest was completely consistent with Example 1, and a composition D1 with antithrombotic function was prepared.

[0037] Comparative Example 2 The raw materials in this comparative composition by weight are: 12 parts mulberry leaves, 8 parts Eucommia ulmoides leaves, 4 parts casein hydrolysate peptides, 5 parts earthworm protein, 10 parts perilla, 7 parts prickly pear, and 1 part nattokinase.

[0038] The difference between this comparative example and Example 1 is that: in step (2), a mixed bacterial solution of Aspergillus oryzae ATCC56747 and Bacillus subtilis ATCC6633 with a volume ratio of 3:1 was used for fermentation. The rest was completely consistent with Example 1, and a composition D2 with antithrombotic function was prepared.

[0039] Comparative Example 3 The raw materials in this comparative composition by weight are: 12 parts mulberry leaves, 8 parts Eucommia ulmoides leaves, 4 parts casein hydrolysate peptides, 5 parts earthworm protein, 10 parts perilla, 7 parts prickly pear, and 1 part nattokinase.

[0040] The difference between this comparative example and Example 1 is that: in step (2), only Aspergillus oryzae ATCC56747 was used for fermentation, while the rest was completely consistent with Example 1, and composition D3 with antithrombotic function was prepared.

[0041] Comparative Example 4 The raw materials in this comparative composition by weight are: 12 parts mulberry leaves, 8 parts Eucommia ulmoides leaves, 4 parts casein hydrolysate peptides, 5 parts earthworm protein, 10 parts perilla, 7 parts prickly pear, and 1 part nattokinase.

[0042] The preparation method of this comparative antithrombotic functional composition includes the following steps: (1) Raw material pretreatment: Take clean dried raw materials of each weight, crush mulberry leaves, Eucommia ulmoides leaves, perilla and prickly pear into powder and pass through a 40-mesh standard sieve, and pass earthworm protein, casein hydrolysate and nattokinase through a 60-mesh sieve. Store in a sealed container at low temperature and away from light for later use.

[0043] (2) Mixed fermentation of mulberry leaves, Eucommia ulmoides leaves, casein hydrolysate peptides, earthworm protein, perilla and prickly pear: After mixing the pretreated mulberry leaves, Eucommia ulmoides leaves, perilla and prickly pear powders, casein hydrolysate peptides and earthworm protein, they were mixed with purified water at a ratio of 1:8 to prepare a suspension; the activated bacterial solutions of Aspergillus oryzae ATCC56747 and Bacillus vesiculus CICC21430 were taken and fermented according to the ratio of Aspergillus oryzae ATCC56747: Bacillus vesiculus CICC21430. The C21430 was mixed at a volume ratio of 3:1 to obtain a composite bacterial solution. The composite bacterial solution was added to the suspension at an inoculum rate of 6% (v / v), and fermented at a constant temperature of 30℃, pH 6.5, and 180r / min for 72h. After fermentation, the bacterial strain was completely inactivated by incubation at 90℃ for 15min. After cooling, the mixture was centrifuged at 8000r / min for 20min. The supernatant was collected and filtered through a 0.22μm ceramic membrane. The filtrate was then freeze-dried under vacuum to obtain extract powder a.

[0044] (3) Mix the composition: Take extract powder a, add 1 part of nattokinase, mix evenly, pass through an 80-mesh sieve to obtain composition D4 with antithrombotic function, and freeze for later use.

[0045] Comparative Example 5 The raw materials in this comparative composition by weight are: 20 parts mulberry leaves, 4 parts casein hydrolysate peptides, 5 parts earthworm protein, 17 parts perilla, and 1 part nattokinase.

[0046] The difference between this comparative example and Example 1 is that the raw materials do not contain Eucommia ulmoides leaves and prickly pear, but are otherwise completely consistent with Example 1, and a composition D5 with antithrombotic function is prepared.

[0047] Comparative Example 6 The raw materials in this comparative composition by weight are: 12 parts mulberry leaves, 8 parts Eucommia ulmoides leaves, 9 parts casein hydrolysate peptides, 10 parts perilla leaves, 7 parts prickly pear, and 1 part nattokinase.

[0048] The difference between this comparative example and Example 1 is that the raw materials do not contain earthworm protein, but are otherwise completely consistent with Example 1, and a composition D6 with antithrombotic function is prepared.

[0049] Comparative Example 7 The raw materials in this comparative composition by weight are: 12 parts mulberry leaves, 8 parts Eucommia ulmoides leaves, 4 parts casein hydrolysate peptides, 6 parts earthworm protein, 10 parts perilla, and 7 parts prickly pear.

[0050] The difference between this comparative example and Example 1 is that the raw materials do not contain nattokinase, but are otherwise completely consistent with Example 1, and a composition D7 with antithrombotic function is prepared.

[0051] Comparative Example 8 The raw materials for this comparative composition by weight are: 20 parts mulberry leaves, 2 parts Eucommia ulmoides leaves, 7 parts casein hydrolysate peptides, 2 parts earthworm protein, 2 parts perilla, 15 parts prickly pear, and 1 part nattokinase. The preparation method of this comparative composition with antithrombotic function is the same as in Example 1, resulting in composition D8 with antithrombotic function.

[0052] Experimental Example 1: Verification of Antithrombotic Activity at the Cellular Level Cell and reagent sources: Human umbilical vein endothelial cells (HUVEC) (model: iCell-h110, Cybio (Shanghai) Biotechnology Co., Ltd.); thrombosis-related markers TXB2 and 6-Keto-PGF1α detection kits (catalog numbers: TXB2-023, KETO-019, Shanghai Yuanye Biotechnology Co., Ltd.).

[0053] Experimental method: Logarithmic growth phase HUVEC cells were seeded in 96-well culture plates, and the cell density was adjusted to 1×10⁶ cells / well. 5 The cells were inoculated at a concentration of 100 μL / mL, and each well was incubated at 37°C in a 5% CO2 incubator for 24 h. The experiment was divided into a blank control group, a model control group, a positive control group, Examples 1-3, and Comparative Examples 1-8, with 6 replicates per group. Except for the blank control group, all other groups used ox-LDL to induce human umbilical vein endothelial cells (HUVECs) to construct a vascular endothelial injury and thrombosis model. After successful modeling, each example and comparative example group was treated with the corresponding composition sample at a final concentration of 100 μg / mL. The positive control group was treated with aspirin at a final concentration of 50 μg / mL, and the blank control group and model control group were treated with an equal volume of basal culture medium. The intervention lasted for 24 h. After the intervention, the levels of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-Keto-PGF1α) in the cell supernatant were detected by enzyme-linked immunosorbent assay (ELISA), and the TXB2 / 6-Keto-PGF1α ratio was calculated to evaluate the regulatory effect of the composition on vascular endothelial thrombosis balance.

[0054] Experimental Results: The results of endothelial function index detection of HUVEC cells in each group are shown in Table 1 below. Experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 29.0 software. One-way ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely statistically significant. Compared with the blank control group, △ indicates P < 0.05, and △△ indicates P < 0.01; compared with the model control group, # indicates P < 0.05, and ## indicates P < 0.01; compared with the Example 1 group, * indicates P < 0.05, and ** indicates P < 0.01; the same applies to the following tables.

[0055] Table 1. Results of endothelial function indicators in HUVEC cells of each group (n=6) Blank control group - 42.63±3.25 17.85±1.22 2.39±0.15 Model control group - <![CDATA[89.65±5.12 △△ ]]> <![CDATA[7.32±0.85 △△ ]]> <![CDATA[12.25±0.68 △△ ]]> Positive control group aspirin <![CDATA[52.42±4.05 ## ]]> <![CDATA[15.56±1.03 ## ]]> <![CDATA[3.37±0.32 ## ]]> Example 1 Group D1 <![CDATA[49.28±3.62 ## ]]> <![CDATA[15.68±1.15 ## ]]> <![CDATA[3.14±0.21 ## ]]> Example 2 group D2 <![CDATA[47.15±3.48 ## ]]> <![CDATA[16.25±1.20 ## ]]> <![CDATA[2.90±0.18 ## ]]> Example 3 Group D3 <![CDATA[52.36±3.85 ## ]]> <![CDATA[14.92±1.08 ## ]]> <![CDATA[3.51±0.25 ## ]]> Comparative Example 1 S1 <![CDATA[65.82±4.25 #* ]]> <![CDATA[10.25±0.92 #* ]]> <![CDATA[6.42±0.38 ##* <!-- 5 -->]]> Comparative Example 2 S2 <![CDATA[67.15±4.36 #* ]]> <![CDATA[9.86±0.88 #* ]]> <![CDATA[6.81±0.42 #* ]]> Comparative Example 3 Groups S3 <![CDATA[70.28±4.52 #* ]]> <![CDATA[9.12±0.82 #* ]]> <![CDATA[7.71±0.45 #** ]]> Comparative Example 4 Groups S4 <![CDATA[76.52±4.85 ** ]]> <![CDATA[8.25±0.76 ** ]]> <![CDATA[9.27±0.52 ** ]]> Comparative Example 5 Groups S5 <![CDATA[82.24±5.26 ** ]]> <![CDATA[7.81±0.68 ** ]]> <![CDATA[10.55±0.65 ** ]]> Comparative Example 6 Groups S6 <![CDATA[82.15±5.32 ** ]]> <![CDATA[8.24±0.66 ** ]]> <![CDATA[9.97±0.66 ** ]]> Comparative Example 7 Groups S7 <![CDATA[78.15±4.92 ** ]]> <![CDATA[9.02±0.73 #** ]]> <![CDATA[8.66±0.56 #** ]]> Comparative Example 8 Groups S8 <![CDATA[79.36±5.01 ** ]]> <![CDATA[7.85±0.71 ** ]]> <![CDATA[10.11±0.59 ** ]]> It can be seen from the cell experiment data in Table 1 that, in ox-LDL-induced model control group, cells showed significant endothelial injury (P<0.01), the prothrombotic factor TXB2 increased abnormally, and the protective factor 6-Keto-PGF1α decreased significantly (P<0.01), and the thrombus homeostasis ratio was greatly imbalanced, indicating that the construction of the vascular endothelial injury thrombus model was successful. Compared with the model control group, the groups of Examples 1 to 3 of the present invention can extremely significantly down-regulate the content of TXB2 and increase the level of 6-Keto-PGF1α (P<0.01), effectively correcting vascular endothelial thrombus disorder, and the effects of endothelial protection and antithrombotic mechanism are significantly better than those of the comparative example groups (P<0.01 or P<0.05). In addition, Comparative Examples 5 and 6, which lack the core components of Cyclocarya paliurus leaves / Rosa roxburghii and earthworm protein, have almost no endothelial repair activity; and it can be seen from the comparison between Comparative Examples 1 to 3 and Example 1 that the microorganisms used in mixed fermentation have a significant impact on the activity of the final composition (P<0.05). It is confirmed from the cell mechanism level that the specific formula and segmented coupling preparation process of the present invention can protect vascular endothelium and inhibit the initial formation of thrombus through multiple targets, and there is a significant synergistic effect between the components and the process. The composition of the present invention can effectively repair vascular endothelial cell injury induced by ox-LDL, down-regulate the expression of prothrombotic factor TXB2, increase the level of vascular protective factor 6-Keto-PGF1α, correct the imbalance of vascular endothelial thrombus homeostasis, inhibit the initiation and formation of thrombus from the cell source, and has excellent pre-regulation effects of vascular endothelial protection and antithrombosis.

[0056] Experiment Example 2: Verification Test of Antithrombotic Activity in Rats

[0057] Animal source: SPF-grade SD rats, male, aged 6-8 weeks, weighing 250-300g, were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd. The production license number of experimental animals is SCXK (Guangdong) 2022-0063, and the quality certificate number is 44829700038486; feeding environment: temperature 22±2°C, humidity 50%±5%, 12h light-dark alternation, free access to food and water, and the experiment was carried out after 7d of adaptive feeding.

[0058] Animal grouping: SD rats were randomly divided into blank control group, model control group, positive control group (aspirin), groups of Examples 1 to 3 and groups of Comparative Examples 1 to 8, with 8 rats in each group. The experiment was started after 3d of adaptive feeding. The blank control group was fed normally without modeling and administration; the other groups were used to construct rat thrombus models by ferric chloride carotid artery thrombosis modeling method. After successful modeling, the positive control group was given aspirin (10mg / kg), each example group and comparative example group were administrated by intragastric gavage at a dose of 100mg / kg, once a day, for consecutive 14d. The blank control group and the model control group were given an equal volume of normal saline by intragastric gavage.

[0059] Detection indicators: 24 hours after the last administration, blood was collected from the abdominal aorta of rats, and plasma was separated by centrifugation. The activated partial thromboplastin time (APTT), prothrombin time (PT), and fibrinolysis rate were detected using the corresponding kits to evaluate the antithrombotic activity of the composition of the present invention.

[0060] Experimental results: The results of the detection of thrombotic activity indicators in rats are shown in Table 2 below. The experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 29.0 software. One-way ANOVA was used for comparison between groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely statistically significant.

[0061] Table 2. Results of the thrombolytic activity of the composition of the present invention in rats (n=8) Blank control group - 38.62±2.15 15.36±0.82 12.35±1.26 Model control group - <![CDATA[22.15±1.86 △△ ]]> <![CDATA[8.24±0.65 △△ ]]> <![CDATA[4.12±0.85 △△ <!-- 6 -->]]> Positive control group aspirin <![CDATA[35.28±2.03 ## ]]> <![CDATA[14.68±0.79 ## ]]> <![CDATA[32.65±2.14 ## ]]> Example 1 Group D1 <![CDATA[36.85±1.92 ## ]]> <![CDATA[13.82±0.76 ## ]]> <![CDATA[29.18±1.96 ## ]]> Example 2 group D2 <![CDATA[37.12±2.05 ## ]]> <![CDATA[13.95±0.81 ## ]]> <![CDATA[28.86±2.03 ## ]]> Example 3 Group D3 <![CDATA[35.96±1.88 ## ]]> <![CDATA[14.06±0.73 ## ]]> <![CDATA[30.52±1.89 ## ]]> Comparative Example 1 S1 <![CDATA[31.25±1.75 # ]]> <![CDATA[11.58±0.68 #* ]]> <![CDATA[22.36±1.65 #* ]]> Comparative Example 2 S2 <![CDATA[30.86±1.82 # ]]> <![CDATA[11.24±0.71 #* ]]> <![CDATA[21.58±1.72 #* ]]> Comparative Example 3 Groups S3 <![CDATA[29.52±1.68 #* ]]> <![CDATA[10.86±0.65 #* ]]> <![CDATA[19.85±1.56 #** ]]> Comparative Example 4 Groups S4 <![CDATA[27.85±1.72 ** ]]> <![CDATA[9.68±0.62 ** ]]> <![CDATA[16.24±1.48 #** ]]> Comparative Example 5 Groups S5 <![CDATA[24.15±1.58 ** ]]> <![CDATA[8.56±0.52 ** ]]> <![CDATA[8.65±1.12 ** ]]> Comparative Example 6 Groups S6 <![CDATA[23.82±1.55 ** ]]> <![CDATA[8.42±0.50 ** ]]> <![CDATA[8.24±1.08 ** ]]> Comparative Example 7 Groups S7 <![CDATA[26.92±1.65 ** ]]> <![CDATA[9.25±0.58 ** ]]> <![CDATA[14.86±1.35 #** ]]> Comparative Example 8 Groups S8 <![CDATA[26.58±1.62 ** ]]> <![CDATA[9.12±0.56 ** ]]> <![CDATA[14.52±1.32 #** ]]> Based on the data in Table 2 regarding the in vivo antithrombotic efficacy of the drug in rats, compared to the blank control group, the coagulation parameters of the rats in the ferric chloride-induced thrombosis model control group were significantly abnormal (P < 0.01), with a substantial decrease in APTT and PT (P < 0.01) and a significant reduction in fibrinolysis rate (P < 0.01). This indicates that the rat thrombosis model was successfully established, and the rats exhibited a hypercoagulable state characterized by hypercoagulability and fibrinolysis inhibition, which can be used to evaluate the in vivo antithrombotic activity of the composition. Furthermore, in the positive control group, aspirin intervention effectively improved the coagulation function of the model rats, significantly prolonging clotting time and enhancing antithrombotic capacity (P < 0.01), confirming that the positive control drug has a clear anticoagulant and thrombolytic effect and can be used as a reference standard for the efficacy of this experiment.

[0062] Animal efficacy experiments showed that, compared with the model control group, after intervention in groups 1-3, the APTT and PT of rats were significantly increased, and the fibrinolysis rate was greatly improved (P<0.01). The improvement effect of each indicator was significantly better than that of the comparative group (P<0.01 or P<0.05), which proved that the raw material ratio and preparation process of the composition specified in this invention can achieve synergistic effect of multiple components and have excellent in vivo anticoagulant and thrombolytic dual activities. Specifically, the antithrombotic indicators of Comparative Examples 1-3, after fermentation with the mixed microbial system replaced, were significantly lower than those of the Example Group (P < 0.05), with shortened coagulation time and decreased fibrinolytic capacity. This indicates that the specific combination of compound microbial strains and the compound fermentation system of this invention are key to achieving efficient conversion of active ingredients in raw materials and enhancing the antithrombotic efficacy of the composition. In addition, Comparative Example 4, which was fermented with all raw materials uniformly, had significantly lower efficacy than the Example Groups (P < 0.01), confirming that the segmented processing technology of this invention can be specifically adapted to the activity release characteristics of different raw materials. The segmented coupling method of low-temperature preservation of heat-sensitive plant components and fermentation activation of protein and peptides can maximize the activity of each component. Comparative Examples 5-7, which lacked the core components of Eucommia ulmoides leaf, prickly pear, earthworm protein, and nattokinase, showed extremely weak improvement in coagulation and fibrinolytic indicators in rats, with most indicators showing no significant difference from the model group. This indicates that each component of this invention is indispensable, and the scientific combination of each raw material component forms a stable synergistic antithrombotic activity.

[0063] The above data analysis shows that the specific raw material formulation ratio, compound microbial fermentation system, and segmented preparation process of the active ingredients of the present invention jointly determine the antithrombotic effect of the composition of the present invention. The elements are mutually compatible and synergistic, and all raw materials of the present invention are food and medicine homologous, green, safe and without side effects. It has extremely high application value and industrialization prospects in the fields of thrombosis prevention, vascular damage repair and post-thrombotic sequelae treatment.

[0064] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A composition having antithrombotic function, characterized in that, The composition is prepared from the following raw materials in parts by weight: 8-16 parts mulberry leaves, 6-10 parts Eucommia ulmoides leaves, 2-6 parts casein hydrolysate peptides, 4-6 parts earthworm protein, 8-12 parts perilla, 5-9 parts prickly pear, and 0.5-2 parts nattokinase.

2. The composition according to claim 1, characterized in that, The composition is prepared from the following raw materials in parts by weight: 12 parts mulberry leaves, 8 parts Eucommia ulmoides leaves, 4 parts casein hydrolysate peptides, 5 parts earthworm protein, 10 parts perilla, 7 parts prickly pear, and 1 part nattokinase.

3. The composition according to any one of claims 1 or 2, characterized in that, The composition also contains pharmaceutically acceptable excipients.

4. The composition according to claim 3, characterized in that, The composition can be prepared into any one of the following dosage forms: powder, granules, tablets, capsules, or oral liquid.

5. A method for preparing a composition with antithrombotic function according to any one of claims 1-4, characterized in that, The process includes the following steps: (1) raw material pretreatment; (2) microbial fermentation to prepare extract powder a; (3) low-temperature ultrasonic synergistic extraction to prepare extract powder b; (4) mixing extract powder a, extract powder b and nattokinase and homogenizing to obtain the composition.

6. The preparation method according to claim 5, characterized in that, The step (1) raw material pretreatment includes: weighing each part by weight of the clean dry raw material of the composition according to any one of claims 1-4, crushing and sieving it and storing it for later use.

7. The preparation method according to claim 5, characterized in that, The step (2) microbial fermentation includes: mixing pretreated mulberry leaves, Eucommia ulmoides leaves, casein hydrolysate peptides, and earthworm protein, and preparing a suspension with purified water at a material-to-liquid ratio of 1:6-10; mixing activated Aspergillus oryzae ATCC56747 and Bacillus vesiculosus CICC21430 activated bacterial solution at a volume ratio of 3:1 to obtain a composite bacterial solution, and adding it to the suspension for fermentation at an inoculum amount of 4-8% v / v.

8. The preparation method according to claim 7, characterized in that, The activated Bacillus vesiculus CICC21430 bacterial solution had a concentration of 1×10⁻⁶. 8 The concentration of the activated Aspergillus oryzae ATCC56747 spore suspension was 1×10 CFU / mL. 7 Spores / mL.

9. The preparation method according to claim 5, characterized in that, The step (3) low-temperature ultrasonic synergistic extraction includes: adding the pretreated perilla and prickly pear powder to a 45% ethanol-purified aqueous solution at a material-liquid ratio of 1:6-10 for ultrasonic extraction.

10. The use of the composition according to any one of claims 1-4 in the preparation of a medicament or health product having antithrombotic function.