A blood-activating and stasis-resolving medicinal composition containing fermented douchi and a preparation method thereof

Abstract

The application provides a blood-activating and stasis-removing medicinal composition containing fermented douchi and a preparation method thereof, belongs to the technical field of biological medicine, and the medicinal composition comprises fermented douchi, drynaria extract, extract of pereskia aculeata, tanshinone II A, notoginsenoside R1, ligustrazine, rhodioside, total flavones of hawthorn leaves and total flavones of mulberry leaves. The fermented douchi is prepared from soybeans and black beans through fermentation by bacillus subtilis and bacillus coagulans. The drynaria extract is prepared from drynaria powder through common enzymolysis by cellulase, chitinase and beta-glucanase, extraction by a two-phase solvent, and purification by a dextran gel column. The extract of pereskia aculeata is prepared from pereskia aculeata powder through common enzymolysis by pectinase, cellulase and xylanase, common enzymolysis by bromelain and subtilisin, and purification by a macroporous adsorption resin column. The medicinal components are prepared in a specific proportion to relieve blood stasis from two dimensions of "improving blood flow dynamics + preventing thrombus formation".

Description

Technical Field

[0001] This invention pertains to the field of biomedical technology, specifically relating to a blood-activating and stasis-removing drug composition containing fermented soybeans and its preparation method. Background Technology

[0002] Blood-activating and stasis-removing drugs are an important category of drugs in the pharmaceutical field that play a therapeutic role in "blood stasis syndrome." Their research and application span the inheritance of traditional medical theories and the innovation of modern pharmaceutical technology, and are one of the typical areas of research combining traditional Chinese and Western medicine. In traditional applications, blood-activating and stasis-removing drugs have long existed in the form of natural medicinal materials and compound prescriptions. The core categories can be divided into three types: First, blood-activating and pain-relieving drugs, such as Chuanxiong and Yanhusuo, relieve pain symptoms such as headache and abdominal pain by clearing blood vessels, and are widely used in chronic pain management; second, blood-activating and menstrual regulating drugs, such as Danggui and Yimucao, are commonly used in gynecology for menstrual disorders and amenorrhea caused by blood stasis in women; and third, blood-breaking and mass-dissolving drugs, such as Sanleng and Ezhu, have strong medicinal effects and are used to treat masses and lumps formed by blood stasis accumulation, and are widely used in traditional adjuvant cancer treatment. These drugs are mostly used in traditional dosage forms such as decoctions and pills, relying on the principle of "treatment based on syndrome differentiation". The combination is adjusted according to the patient's constitution and blood stasis syndrome, and rich application experience has been accumulated in thousands of years of clinical practice.

[0003] With the development of modern medical technology, drugs for promoting blood circulation and removing blood stasis have entered a modern research and development stage. From the mid-20th century, researchers began to isolate and identify the effective components of traditional drugs, gradually clarifying core pharmacological substances such as tanshinone IIA in Danshen, notoginsenoside R1 in Sanqi, and ligustrazine in Chuanxiong. These components have become key targets for modern formulation development. In terms of dosage form innovation, traditional prescriptions have broken through the limitations of decoctions, developing modern dosage forms such as injections (e.g., Danshen injection), tablets (e.g., compound Danshen tablets), and pills (e.g., Danshen pills), significantly improving drug absorption efficiency and ease of use, meeting the needs of different clinical scenarios (e.g., injections for emergency treatment of acute cardiovascular and cerebrovascular diseases, and pills for long-term management of chronic diseases).

[0004] In the field of pharmacological mechanism research, modern medicine has provided scientific support for drugs that promote blood circulation and remove blood stasis. Studies have confirmed that these drugs work through multiple targets: first, they improve blood circulation, dilate blood vessels, reduce blood viscosity, and increase tissue blood supply, making them suitable for ischemic diseases such as coronary heart disease and cerebral infarction; second, they inhibit platelet aggregation, reduce thrombus formation, and lower the risk of thrombotic diseases; third, they regulate coagulation function, balance the coagulation and fibrinolytic systems, and prevent bleeding or thrombosis tendencies; and fourth, they promote tissue repair and accelerate the absorption of blood stasis, showing significant effects in scenarios such as traumatic injuries and postoperative recovery. The clarification of these mechanisms has driven the transformation of drugs that promote blood circulation and remove blood stasis from "empirical medication" to "evidence-based medication," and some drugs have passed international multi-center clinical trials, becoming important options for the integrated treatment of cardiovascular and cerebrovascular diseases and orthopedic diseases using traditional Chinese and Western medicine.

[0005] Currently, there are various blood-activating and stasis-removing drugs on the market, but their quality varies greatly. Some drugs pose a risk of side effects with long-term use, while others have the problem of long-term effects but unclear results. Therefore, the research and development of blood-activating and stasis-removing drugs is still ongoing, aiming to develop safer and more effective treatments to provide better therapeutic outcomes for patients with blood stasis. Summary of the Invention

[0006] To address the issues of side effects, long treatment duration, and limited efficacy associated with existing blood-activating and stasis-removing drugs, this invention provides a blood-activating and stasis-removing drug composition containing fermented soybeans and its preparation method. The composition utilizes a special method to prepare fermented soybeans, *Gnaphalium affine* extract, and *Akebia quinata* peel extract, combined with tanshinone IIA, notoginsenoside R1, ligustrazine, rhodioloside, total flavonoids from hawthorn leaves, and total flavonoids from mulberry leaves in specific proportions to create a drug combination. This combination alleviates blood stasis from two dimensions: improving hemodynamics and preventing thrombosis, by dilating blood vessels, reducing blood viscosity, inhibiting platelet aggregation, and balancing the coagulation-fibrinolytic system. This aligns with the core pathological mechanism of "ischemia + thrombosis" in cardiovascular and cerebrovascular diseases, and is used for the effective prevention and treatment of blood stasis and cardiovascular and cerebrovascular diseases. The specific technical solution is as follows:

[0007] A blood-activating and stasis-removing drug composition containing fermented soybeans is composed of the following raw materials in parts by weight: 30-40 parts fermented soybeans, 15-20 parts *Gynostemma pentaphyllum* extract, 15-20 parts *Akebia quinata* peel extract, 10-15 parts tanshinone IIA, 8-12 parts notoginsenoside R1, 5-10 parts ligustrazine, 3-5 parts rhodioloside, 3-4 parts total flavonoids from hawthorn leaves, and 1-2 parts total flavonoids from mulberry leaves.

[0008] The fermented soybean paste is made from soybeans and black beans through fermentation with Bacillus subtilis and Bacillus coagulans. Specifically, the preparation of the fermented soybean paste includes: soybeans and black beans in a mass ratio of 1:(0.4-0.6) are steamed and sterilized, and then fermented sequentially with Bacillus subtilis and Bacillus coagulans to obtain the product.

[0009] The *Gynostemma pentaphyllum* extract is prepared by enzymatic hydrolysis of *Gynostemma pentaphyllum* powder with cellulase, chitinase, and β-glucanase, followed by biphasic solvent extraction and purification using a Sephadex LH-20 dextran gel column. Specifically, the preparation of the *Gynostemma pentaphyllum* extract includes: enzymatic hydrolysis of *Gynostemma pentaphyllum* powder with cellulase, chitinase, and β-glucanase to obtain enzymatically hydrolyzed bacterial powder, followed by extraction with a biphasic solvent prepared with ethanol, water, and ammonium sulfate; the upper phase is collected and subjected to a Sephadex LH-20 dextran gel column, successively washed with deionized water, 30%–35% (v / v) ethanol aqueous solution, and 50%–55% (v / v) ethanol aqueous solution to remove impurities; eluted with 70%–75% (v / v) ethanol aqueous solution; the eluent is collected, concentrated under reduced pressure, and freeze-dried to obtain the product.

[0010] The August melon rind extract is prepared by enzymatic hydrolysis of August melon rind powder by pectinase, cellulase, and xylanase, followed by enzymatic hydrolysis by bromelain and subtilisin, and purification via AB-8 macroporous adsorption resin column. Specifically, the preparation of the August melon rind extract includes: washing August melon rind powder with sodium bicarbonate aqueous solution, followed by enzymatic hydrolysis by pectinase, cellulase, and xylanase, then adding the enzymatic hydrolysate obtained by enzymatic hydrolysis with bromelain and subtilisin, centrifuging, collecting the supernatant, concentrating under reduced pressure, loading the sample onto an AB-8 macroporous adsorption resin column, first rinsing with deionized water to remove impurities, then eluting with 50%–60% ethanol aqueous solution for 4–5 column volumes, collecting the eluent, concentrating under reduced pressure, and freeze-drying to obtain the product.

[0011] The preparation method of the fermented soybeans in the above-mentioned pharmaceutical composition includes: steaming and sterilizing soybeans, cooling to 35℃~38℃, adding sterile water, inoculating with Bacillus subtilis activated bacterial solution under stirring, and performing aerobic fermentation at 35℃~38℃ for 20h~24h, sterilizing by heating, cooling to 40℃~45℃, inoculating with Bacillus coagulans activated bacterial solution under stirring, and performing aerobic fermentation at 40℃~45℃ for 16h~18h; sterilizing by heating, drying, and pulverizing into powder to obtain fermented soybeans.

[0012] In the above-mentioned method for preparing fermented soybeans, the soybeans are yellow soybeans and black soybeans in a mass ratio of 1:(0.4-0.6); the soybeans are soaked and drained before steaming and sterilization; the steaming and sterilization is carried out at a temperature of 115℃-122℃ and a steam pressure of 0.12MPa-0.14MPa for 40-50 minutes; the amount of sterile water used is 30%-35% of the soybean mass; the inoculation amount of Bacillus subtilis activated bacterial solution is 4%-6% of the soybean mass; and the concentration of the Bacillus subtilis activated bacterial solution is 1.0×10⁻⁶. 9 CFU / mL ~5.0×10 9 CFU / mL; the inoculation amount of the activated Bacillus coagulans solution is 2%–3% of the soybean mass; the concentration of the activated Bacillus coagulans solution is 5.0 × 10⁻⁶ CFU / mL. 8 CFU / mL ~ 2.0 × 10 9 CFU / mL; the temperature sterilization was carried out at 80℃~90℃ for 10min~20min; the drying was carried out at 50℃~60℃ until the moisture content was less than 5wt%.

[0013] The preparation method of the *Gynostemma pentaphyllum* extract in the above-mentioned pharmaceutical composition includes: adding citrate-disodium hydrogen phosphate buffer to *Gynostemma pentaphyllum* powder, then adding cellulase, chitinase, and β-glucanase, stirring and hydrolyzing at 45℃~50℃ for 2h~3h, then adding fig protease, stirring and hydrolyzing at 60℃~65℃ for 1.5h~2h, inactivating the enzyme by heating, concentrating under reduced pressure, and freeze-drying to obtain enzymatically hydrolyzed bacterial powder; preparing a biphasic solvent according to the mass ratio of ethanol:water:ammonium sulfate = (25~30):(70~75):(1~1.5); adding the enzymatically hydrolyzed bacterial powder to the biphasic solvent according to the material-liquid ratio of 1g:12mL~1g:15mL, adding 0.1%~0.2% L-ascorbic acid by mass of the enzymatically hydrolyzed bacterial powder, sonicating, allowing to stand and separate into layers, taking the upper phase, and loading the sample onto Sephadex. The LH-20 dextran gel column was eluted sequentially with one column volume each of deionized water, 30%–35% ethanol aqueous solution, and 50%–55% ethanol aqueous solution to remove impurities. Finally, it was eluted with 70%–75% ethanol aqueous solution for 2–3 column volumes. The eluent was collected, concentrated under reduced pressure to remove ethanol, and freeze-dried to obtain the *Bacillus thuringiensis* extract.

[0014] In the above-mentioned method for preparing the extract of *Clerodendrum trichotomum*, the *Clerodendrum trichotomum* powder is obtained by drying *Clerodendrum trichotomum* below 60°C, pulverizing it, and passing it through a 200-250 mesh sieve; the amount of citrate-disodium hydrogen phosphate buffer added is 6-8 times the mass of the *Clerodendrum trichotomum* powder; the pH value of the citrate-disodium hydrogen phosphate buffer is 5.0-5.5; the amount of cellulase, chitinase, and β-glucanase added is 0.5%-1% of the mass of the *Clerodendrum trichotomum* powder; the amount of fig protease added is 0.8%-1.5% of the mass of the *Clerodendrum trichotomum* powder; the stirring speed for enzymatic hydrolysis is 50-80 rpm; the enzyme inactivation by heating is to 85-90°C for 10-15 minutes; the ultrasonic treatment is to ultrasonically treat at 50-55°C and 300-350W power for 30-60 minutes; and the temperature for vacuum concentration is 50-60°C.

[0015] The preparation method of the August melon peel extract in the above-mentioned pharmaceutical composition includes: adding August melon peel powder to a sodium bicarbonate aqueous solution, stirring, centrifuging, and collecting the precipitate; adding 8 to 12 times the mass of water to adjust the pH to 4.5 to 5.0, then adding pectinase, cellulase, and xylanase, and performing a first enzymatic hydrolysis at 50℃ to 55℃ and 50 rpm to 80 rpm for 2 to 3 hours, adjusting the pH to 6.5 to 7.5, adding bromelain and subtilisin, and performing a second enzymatic hydrolysis at 50℃ to 55℃ and 50 rpm to 80 rpm for 1 to 2 hours, heating to inactivate the enzymes, cooling, centrifuging, and collecting the supernatant; concentrating the supernatant under reduced pressure, loading it onto an AB-8 macroporous adsorption resin column, first rinsing with deionized water for 2 to 3 column volumes to remove impurities, then eluting with 50% to 60% volume concentration ethanol aqueous solution for 4 to 5 column volumes, collecting the eluent, concentrating under reduced pressure to remove ethanol, and freeze-drying to obtain the August melon peel extract.

[0016] In the above-mentioned preparation method of the August melon rind extract, the August melon rind powder is obtained by taking the rind of August melons that are 80% ripe or more, drying them below 60℃, pulverizing them, and passing them through a 200-250 mesh sieve; the amount of sodium bicarbonate aqueous solution used is 4 to 6 times the mass of the August melon rind powder; the concentration of the sodium bicarbonate aqueous solution is 0.5 wt% to 0.8 wt%; the stirring is carried out at 40℃ to 45℃ and 50 rpm to 80 rpm for 30 to 60 minutes; the fruit... The amounts of gelase, cellulase, and xylanase added are all 0.5% to 1.5% of the weight of the Akebia quinata rind powder; the amounts of bromelain and subtilisin added are all 0.5% to 1.5% of the weight of the Akebia quinata rind powder; the enzyme inactivation is carried out by heating to 85℃ to 90℃ for 10 to 15 minutes; the centrifugation is carried out at a speed of 6000 rpm to 8000 rpm for 10 to 15 minutes; the temperature for vacuum concentration is 50℃ to 60℃.

[0017] The preparation method of the above-mentioned blood-activating and stasis-removing drug composition containing fermented soybeans includes the following steps: mixing the prescribed amounts of fermented soybeans, extract of dried mushroom, extract of akebia quinata peel, tanshinone IIA, notoginsenoside R1, ligustrazine, rhodioloside, total flavonoids of hawthorn leaves and total flavonoids of mulberry leaves evenly to obtain the drug composition.

[0018] The above-mentioned pharmaceutical composition is mixed with pharmaceutically acceptable excipients to prepare any pharmaceutically acceptable dosage form, including tablets, capsules, granules, pills, powders, and oral liquids.

[0019] The use of the above-mentioned pharmaceutical composition in the preparation of a medicine for the prevention and / or treatment of blood stasis syndrome and cardiovascular and cerebrovascular diseases.

[0020] This invention provides a blood-activating and stasis-removing pharmaceutical composition containing fermented soybeans and its preparation method, the beneficial effects of which include:

[0021] I. The drug of this invention can safely and effectively improve blood stasis syndrome and related pathological conditions of cardiovascular and cerebrovascular diseases. The core principle is as follows:

[0022] 1. Multi-target regulation of blood circulation: By dilating blood vessels, reducing blood viscosity, inhibiting platelet aggregation, and balancing the coagulation-fibrinolysis system, it alleviates blood stasis from the dual dimensions of "improving hemodynamics + preventing thrombosis", which is in line with the core pathological mechanism of "ischemia + thrombosis" in cardiovascular and cerebrovascular diseases.

[0023] 2. Synergistic protection of vascular endothelium: The flavonoids and polysaccharides in the drug can scavenge free radicals, inhibit inflammatory responses, stabilize the structure and function of vascular endothelial cells, reduce the "pathological basis" of platelet activation and adhesion, and create a favorable microenvironment for the blood-activating components to exert their effects.

[0024] 3. High safety: Acute toxicity tests and long-term toxicity tests show that the drug has no obvious toxicity. Its raw materials are mostly natural fermentation and extraction products, and the active ingredients are precisely purified, which reduces the risk of "complex ingredients leading to side effects" in traditional drugs.

[0025] II. In the preparation of fermented soybean paste, soybeans and black beans are mixed in a specific ratio: the two types of soybeans complement each other, providing a comprehensive substrate for fermentation and ensuring the diversity of active peptides and amino acids. Fermentation is carried out in stages using Bacillus subtilis and Bacillus coagulans: the former produces proteases and cellulases to break down macromolecules, while the latter produces lactic acid and antimicrobial peptides, which not only optimizes the flavor but also enriches the spectrum of active ingredients, improving drug safety and bioavailability.

[0026] III. In the preparation of *Gynostemma pentaphyllum* extract, a complex enzymatic hydrolysis process involving cellulase, chitinase, β-glucanase, and fig protease is employed: This first breaks down the tough cell wall of *Gynostemma pentaphyllum* (containing cellulose and chitin), then hydrolyzes intracellular proteins, fully releasing polysaccharides, functional peptides, and other active ingredients, thus enhancing the extract's activity. Two-phase solvent ultrasonic extraction: Two-phase solvents can directionally extract polar and non-polar active ingredients, while ultrasound assistance improves extraction efficiency and reduces the loss of active ingredients. Sephadex LH-20 column (deionized water + gradient ethanol elution): This precisely separates small-molecule active ingredients (such as flavonoids and short peptides) and removes large-molecule impurities (such as starch and crude fiber), improving extract purity and enhancing efficacy.

[0027] IV. In the preparation of Akebia quinata rind extract, washing with sodium bicarbonate aqueous solution removes acidic impurities and pigments from the surface of the rind, reducing interference from inactive components in subsequent extraction. Pectinase + cellulase + xylanase + bromelain + subtilisin: This process enzymatically hydrolyzes pectin, cellulose, and protein stepwise, breaking down the component complex and releasing active substances such as flavonoids, polysaccharides, and bioactive peptides, thus improving the extract's activity. AB-8 macroporous adsorption resin: AB-8's weakly polar properties specifically adsorb moderately polar flavonoids, bioactive peptides, and other components, efficiently removing impurities and increasing the content of effective components in the extract.

[0028] V. The nine ingredients in the drug formulation form a highly efficient blood-activating system through "functional complementarity and synergistic mechanism." The core synergistic principle is as follows:

[0029] 1. Synergistic effect of core blood-activating components: Tanshinone IIA (calcium channel blockade, antioxidant), Panax notoginseng saponin R1 (anticoagulation, protection of vascular endothelium), and Ligusticum chuanxiong (vasodilator, antiplatelet) complement each other's targets. Tanshinone IIA improves vascular smooth muscle contraction, Panax notoginseng saponin R1 regulates the coagulation system, and Ligusticum chuanxiong promotes hemodynamics. Together, they enhance the core efficacy of "improving circulation + preventing thrombosis" and avoid the shortcomings of single components with "limited targets".

[0030] 2. Synergy between natural extracts and core ingredients: The active peptides and amino acids in fermented soybeans, the polysaccharides and active peptides in dried mushroom extract, and the flavonoids, polysaccharides, and active peptides in Akebia quinata peel extract can assist the core ingredients in three ways: First, by enhancing the bioavailability of the core ingredients (e.g., peptides promote the intestinal absorption of saponins); second, by improving the pathological microenvironment (e.g., polysaccharides have anti-inflammatory properties, flavonoids have antioxidant properties, and they reduce vascular endothelial damage); and third, by supplementing the effects not covered by the core ingredients (e.g., active peptides in fermented soybeans regulate blood lipids and help reduce blood viscosity).

[0031] 3. Synergistic effect of flavonoids: Hawthorn leaf total flavonoids (enhancing the lipolytic effect of tanshinone IIA) and mulberry leaf total flavonoids (antioxidant and anti-inflammatory) are formulated in a specific ratio to form a highly efficient synergistic effect of "antioxidant-lipolysis", which enhances its vascular protection effect. As core blood-activating components, they "protect" each other and amplify the overall efficacy. Detailed Implementation

[0032] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0033] Example 1

[0034] A blood-activating and stasis-removing drug composition containing fermented soybeans is composed of the following raw materials in parts by weight: 35 parts fermented soybeans, 18 parts *Gynostemma pentaphyllum* extract, 16 parts *Akebia quinata* peel extract, 12 parts tanshinone IIA, 10 parts notoginsenoside R1, 8 parts ligustrazine, 4 parts rhodioloside, 3.5 parts total flavonoids from hawthorn leaves, and 1.5 parts total flavonoids from mulberry leaves.

[0035] The preparation methods for each extract are as follows:

[0036] The preparation of fermented soybean paste includes the following steps: Weigh soybeans and black beans in a mass ratio of 1:0.5, mix them evenly, soak them, and drain. Sterilize the soaked soybeans by steaming them at 120℃ and 0.13MPa for 45 minutes. Cool the steamed soybeans to 37℃, add 32% (by weight of the soybeans) of sterile water, and inoculate with 5% (by weight of the soybeans) of activated Bacillus subtilis solution (concentration 3.0 × 10⁻⁶). 9 The sample was fermented aerobicly at 37°C for 22 hours (CFU / mL). After the first stage of fermentation, it was sterilized at 85°C for 15 minutes. The temperature was then lowered to 42°C, and the sample was inoculated with 2.5% (by weight of soybean) of activated Bacillus coagulans solution (concentration 1.0 × 10⁻⁶ CFU / mL) while stirring. 9 The product (CFU / mL) was subjected to aerobic fermentation at 42℃ for 17 hours. After the second stage of fermentation, it was sterilized at 85℃ for 15 minutes. The fermentation product was dried at 55℃ to a moisture content of 4.6wt%, and finally pulverized into powder to obtain fermented black soybeans.

[0037] The preparation of *Clerodendrum chinense* extract includes the following steps: *Clerodendrum chinense* is dried at 55℃, pulverized, and passed through a 200-mesh sieve to obtain *Clerodendrum chinense* powder. Seven times the mass of a citrate-disodium hydrogen phosphate buffer solution (pH 5.2) is added to the *Clerodendrum chinense* powder, followed by 0.8% (by mass) of cellulase, 0.8% (by mass) of chitinase, and 0.6% (by mass) of β-glucanase. Enzymatic hydrolysis is carried out at 48℃ and 60 rpm for 2.5 h with stirring. Then, 1.0% (by mass) of fig protease is added, and enzymatic hydrolysis is carried out at 62℃ and 60 rpm for 1.5 h with stirring. After enzymatic hydrolysis, the temperature is raised to 88℃ and held for 12 min to inactivate the enzymes. The enzyme-inactivated hydrolysate is concentrated under reduced pressure at 55℃ and then freeze-dried to obtain the enzymatically hydrolyzed bacterial powder. A two-phase solvent is prepared at a mass ratio of ethanol:water:ammonium sulfate = 28:73:1.2. The enzymatically hydrolyzed bacterial powder was added to a biphasic solvent at a material-to-liquid ratio of 1 g:13 mL, and 0.15% L-ascorbic acid (by weight of the enzymatically hydrolyzed bacterial powder) was added as an antioxidant. The mixture was sonicated at 53℃ and 300W for 45 min, then allowed to stand for separation. The upper phase (ethanol-partial water) was collected and loaded onto a Sephadex LH-20 dextran gel column. The column was eluted sequentially with deionized water, 32% (v / v) ethanol-water solution, and 52% (v / v) ethanol-water solution for one column volume each to remove impurities. Finally, it was eluted with 73% (v / v) ethanol-water solution for 2.5 column volumes, and this eluent was collected. The eluent was concentrated under reduced pressure at 55℃ to remove ethanol, and then freeze-dried to obtain the *Bacillus thuringiensis* extract.

[0038] The preparation of the August melon rind extract includes the following steps: August melon rinds that are 80% ripe or more are dried at 55℃, pulverized, and passed through a 200-mesh sieve to obtain August melon rind powder. The August melon rind powder is added to 5 times its weight of a 0.6wt% sodium bicarbonate aqueous solution and stirred at 42℃ and 60rpm for 45 minutes, then centrifuged to collect the precipitate. The precipitate is added to 10 times its weight of water to adjust the pH to 4.8. Then, 1.0% (by weight) of pectinase, 1.0% (by weight) of cellulase, and 1.0% (by weight) of xylanase are added to the August melon rind powder, and the mixture is stirred at 52℃ and 60rpm for a first enzymatic hydrolysis for 2.5 hours. After this, the pH is adjusted to 7.0, and 1.0% (by weight) of bromelain and 1.2% (by weight) of subtilisin are added to the August melon rind powder, and the mixture is stirred at 52℃ and 60rpm for a second enzymatic hydrolysis for 1.5 hours. After enzymatic hydrolysis, the temperature was raised to 88℃ and held for 12 minutes to inactivate the enzyme. After cooling, the sample was centrifuged at 7000 rpm for 12 minutes, and the supernatant was collected. The supernatant was concentrated under reduced pressure at 55℃ and then loaded onto an AB-8 macroporous adsorption resin column. The column was first washed with deionized water for 2.5 column volumes to remove impurities, and then eluted with 55% (v / v) ethanol aqueous solution for 4.5 column volumes. The eluent was collected. The eluent was concentrated under reduced pressure at 55℃ to remove ethanol, and then freeze-dried to obtain the akebia quinata peel extract.

[0039] The preparation method of the pharmaceutical composition includes the following steps: mixing fermented soybeans, *Gnaphalium affine* extract, *Akebia quinata* peel extract, tanshinone IIA, notoginsenoside R1, ligustrazine, rhodioloside, total flavonoids from hawthorn leaves, and total flavonoids from mulberry leaves in the prescribed amounts until homogeneous to obtain the pharmaceutical composition.

[0040] Example 2

[0041] A blood-activating and stasis-removing drug composition containing fermented soybeans is composed of the following raw materials in parts by weight: 30 parts fermented soybeans, 20 parts dried mushroom extract, 15 parts Akebia quinata peel extract, 15 parts tanshinone IIA, 8 parts notoginseng saponin R1, 10 parts ligustrazine, 3 parts rhodioloside, 4 parts total flavonoids from hawthorn leaves and 1 part total flavonoids from mulberry leaves.

[0042] The preparation methods for each extract are as follows:

[0043] The preparation of fermented soybean paste includes the following steps: Weigh soybeans and black beans in a mass ratio of 1:0.6, mix them evenly, soak them, and drain. Sterilize the soaked soybeans by steaming them at 115℃ and 0.14MPa for 40 minutes. Cool the steamed soybeans to 38℃, add 30% (by weight of the soybeans) of sterile water, and inoculate with 6% (by weight of the soybeans) of activated Bacillus subtilis solution (concentration 1.0 × 10⁻⁶). 9The sample was fermented aerobicly at 38℃ for 20 hours (CFU / mL). After the first stage of fermentation, it was sterilized at 90℃ for 10 minutes. The temperature was then lowered to 40℃, and the sample was inoculated with 2% (by weight of soybean) of activated Bacillus coagulans solution (concentration 2.0 × 10⁻⁶ CFU / mL) while stirring. 9 The product (CFU / mL) was subjected to aerobic fermentation at 40℃ for 18 hours. After the second stage of fermentation, it was sterilized at 80℃ for 20 minutes. The fermentation product was dried at 50℃ to a moisture content of 4.8wt%, and finally pulverized into powder to obtain fermented black soybeans.

[0044] The preparation of *Clerodendrum chinense* extract includes the following steps: *Clerodendrum chinense* is dried at 50℃, pulverized, and passed through a 250-mesh sieve to obtain *Clerodendrum chinense* powder. Six times the mass of a citrate-disodium hydrogen phosphate buffer solution (pH 5.5) is added to the *Clerodendrum chinense* powder, followed by 0.5% (by mass) cellulase, 1% (by mass) chitinase, and 0.5% (by mass) β-glucanase. Enzymatic hydrolysis is carried out at 50℃ and 50 rpm for 3 hours. Then, 0.8% (by mass) fig protease is added, and enzymatic hydrolysis is carried out at 65℃ and 50 rpm for 2 hours. After enzymatic hydrolysis, the temperature is raised to 85℃ and held for 15 minutes to inactivate the enzymes. The enzyme-inactivated hydrolysate is concentrated under reduced pressure at 50℃ and then freeze-dried to obtain the enzymatically hydrolyzed bacterial powder. A two-phase solvent is prepared at a mass ratio of ethanol:water:ammonium sulfate = 30:70:1.5. The enzymatically hydrolyzed bacterial powder was added to a biphasic solvent at a material-to-liquid ratio of 1 g:12 mL, and 0.2% L-ascorbic acid (by weight of the bacterial powder) was added as an antioxidant. The mixture was sonicated at 50℃ and 350W for 30 min, then allowed to stand for separation. The upper phase (ethanol-partial water) was collected and loaded onto a Sephadex LH-20 dextran gel column. The column was eluted sequentially with deionized water, 35% ethanol aqueous solution, and 50% ethanol aqueous solution for one column volume each to remove impurities. Finally, it was eluted with 75% ethanol aqueous solution for two column volumes, and the eluent was collected. The eluent was concentrated under reduced pressure at 60℃ to remove ethanol, and then freeze-dried to obtain the *Bacillus thuringiensis* extract.

[0045] The preparation of the August melon rind extract includes the following steps: Take August melon rinds that are 80% ripe or more, dry them at 50℃, pulverize them, and pass them through a 250-mesh sieve to obtain August melon rind powder. Add the August melon rind powder to 4 times its weight of a 0.8wt% sodium bicarbonate aqueous solution, stir at 40℃ and 80rpm for 30min, then centrifuge and collect the precipitate. Add the precipitate to 12 times its weight of water, adjust the pH to 4.5, then add 1.5% (by weight) of pectinase, 0.5% (by weight) of cellulase, and 1.5% (by weight) of xylanase from the August melon rind powder, and perform a first enzymatic hydrolysis at 50℃ and 80rpm for 2h. After completion, adjust the pH to 7.5, add 0.5% (by weight) of bromelain and 1.5% (by weight) of Bacillus subtilis protease from the August melon rind powder, and perform a second enzymatic hydrolysis at 50℃ and 80rpm for 1h. After enzymatic hydrolysis, the temperature was raised to 90℃ and held for 10 minutes to inactivate the enzyme. After cooling, the sample was centrifuged at 6000 rpm for 15 minutes, and the supernatant was collected. The supernatant was concentrated under reduced pressure at 50℃ and then loaded onto an AB-8 macroporous adsorption resin column. The column was first washed with deionized water for 3 column volumes to remove impurities, and then eluted with 50% (v / v) ethanol aqueous solution for 5 column volumes. The eluent was collected. The eluent was concentrated under reduced pressure at 50℃ to remove ethanol, and then freeze-dried to obtain the akebia quinata rind extract.

[0046] The preparation method of the pharmaceutical composition includes the following steps: mixing fermented soybeans, *Gnaphalium affine* extract, *Akebia quinata* peel extract, tanshinone IIA, notoginsenoside R1, ligustrazine, rhodioloside, total flavonoids from hawthorn leaves, and total flavonoids from mulberry leaves in the prescribed amounts until homogeneous to obtain the pharmaceutical composition.

[0047] Example 3

[0048] A blood-activating and stasis-removing drug composition containing fermented soybeans is composed of the following raw materials in parts by weight: 40 parts fermented soybeans, 15 parts dried mushroom extract, 20 parts Akebia quinata peel extract, 10 parts tanshinone IIA, 12 parts notoginsenoside R1, 5 parts ligustrazine, 5 parts rhodioloside, 3 parts total flavonoids from hawthorn leaves and 2 parts total flavonoids from mulberry leaves.

[0049] The preparation methods for each extract are as follows:

[0050] The preparation of fermented soybean paste includes the following steps: Weigh soybeans and black beans in a mass ratio of 1:0.4, mix them evenly, and then soak and drain. Sterilize the soaked soybeans by steaming them at 122℃ and 0.12MPa for 50 minutes. Cool the steamed soybeans to 35℃, add 35% (by weight of the soybeans) of sterile water, and inoculate with 4% (by weight of the soybeans) of activated Bacillus subtilis solution (concentration 5.0 × 10⁻⁶). 9The sample was fermented aerobicly at 35°C for 24 hours (CFU / mL). After the first stage of fermentation, it was sterilized at 80°C for 20 minutes. The temperature was then lowered to 45°C, and the sample was inoculated with 3% (by weight of soybean) of activated Bacillus coagulans solution (concentration 5.0 × 10⁻⁶ CFU / mL) while stirring. 8 The product (CFU / mL) was subjected to aerobic fermentation at 45℃ for 16 hours. After the second stage of fermentation, it was sterilized at 90℃ for 10 minutes. The fermentation product was dried at 60℃ to a moisture content of 3.5wt%, and finally pulverized into powder to obtain fermented black soybeans.

[0051] The preparation of *Clerodendrum chinense* extract includes the following steps: *Clerodendrum chinense* is dried at 58℃, pulverized, and passed through a 200-mesh sieve to obtain *Clerodendrum chinense* powder. Eight times the mass of a citrate-disodium hydrogen phosphate buffer solution (pH 5.0) is added to the *Clerodendrum chinense* powder, followed by 1% (by mass) cellulase, 0.5% (by mass) chitinase, and 1% (by mass) β-glucanase. Enzymatic hydrolysis is carried out at 45℃ and 80 rpm for 2 hours. Then, 1.5% (by mass) fig protease is added, and enzymatic hydrolysis is carried out at 60℃ and 80 rpm for 1.5 hours. After enzymatic hydrolysis, the temperature is raised to 90℃ and held for 10 minutes to inactivate the enzymes. The enzyme-inactivated hydrolysate is concentrated under reduced pressure at 60℃ and then freeze-dried to obtain the enzymatically hydrolyzed bacterial powder. A two-phase solvent is prepared at a mass ratio of ethanol:water:ammonium sulfate = 25:75:1. The enzymatically hydrolyzed bacterial powder was added to a biphasic solvent at a material-to-liquid ratio of 1 g:15 mL, and 0.1% L-ascorbic acid (by weight of the enzymatically hydrolyzed bacterial powder) was added as an antioxidant. The mixture was sonicated at 55℃ and 300W for 60 min, then allowed to stand for separation. The upper phase (ethanol-partial water) was collected and loaded onto a Sephadex LH-20 dextran gel column. The column was eluted sequentially with deionized water, 30% ethanol aqueous solution, and 55% ethanol aqueous solution for one column volume each to remove impurities. Finally, it was eluted with 70% ethanol aqueous solution for three column volumes, and the eluent was collected. The eluent was concentrated under reduced pressure at 50℃ to remove ethanol, and then freeze-dried to obtain the *Bacillus thuringiensis* extract.

[0052] The preparation of the August melon rind extract includes the following steps: August melon rinds that are 80% ripe or more are dried at 58℃, pulverized, and passed through a 200-mesh sieve to obtain August melon rind powder. The August melon rind powder is added to 6 times its weight of a 0.5wt% sodium bicarbonate aqueous solution and stirred at 45℃ and 50rpm for 60 minutes, then centrifuged to collect the precipitate. The precipitate is added to 8 times its weight of water to adjust the pH to 5.0. Then, 0.5% (by weight of the August melon rind powder) of pectinase, 1.5% (by weight of the August melon rind powder) of cellulase, and 0.5% (by weight of the August melon rind powder) of xylanase are added, and the mixture is stirred at 55℃ and 50rpm for a first enzymatic hydrolysis for 3 hours. After completion, the pH is adjusted to 6.5, and 1.5% (by weight of the August melon rind powder) of bromelain and 0.5% (by weight of the August melon rind powder) of Bacillus subtilis protease are added, and the mixture is stirred at 55℃ and 50rpm for a second enzymatic hydrolysis for 2 hours. After enzymatic hydrolysis, the temperature was raised to 85℃ and held for 15 minutes to inactivate the enzyme. After cooling, the sample was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. The supernatant was concentrated under reduced pressure at 60℃ and then loaded onto an AB-8 macroporous adsorption resin column. The column was first washed with deionized water for two column volumes to remove impurities, and then eluted with 60% (v / v) ethanol aqueous solution for four column volumes. The eluent was collected. The eluent was concentrated under reduced pressure at 60℃ to remove ethanol, and then freeze-dried to obtain the akebia quinata peel extract.

[0053] The preparation method of the pharmaceutical composition includes the following steps: mixing fermented soybeans, *Gnaphalium affine* extract, *Akebia quinata* peel extract, tanshinone IIA, notoginsenoside R1, ligustrazine, rhodioloside, total flavonoids from hawthorn leaves, and total flavonoids from mulberry leaves in the prescribed amounts until homogeneous to obtain the pharmaceutical composition.

[0054] The pharmaceutical compositions prepared in the above embodiments are mixed with pharmaceutically acceptable excipients to prepare any pharmaceutically acceptable dosage form, including tablets, capsules, granules, pills, powders, and oral liquids. Use of the pharmaceutical compositions in the preparation of remedies for the prevention and / or treatment of blood stasis syndrome and cardiovascular and cerebrovascular diseases.

[0055] The raw materials used in the above embodiments are sourced as follows: Bacillus subtilis from Hubei Rishengchang New Material Technology Co., Ltd.; Bacillus coagulans from Anhui Zhonghong Bioengineering Co., Ltd.; cellulase with an activity of 100,000 / g from Nanjing Jingchang Biotechnology Co., Ltd.; chitinase with an activity of 100,000 / g from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.; β-glucanase with an activity of 100,000 / g from Guangdong Osman Biotechnology Co., Ltd.; fig protease with an activity of 100,000 / g from Shandong Yiwei Bioengineering Co., Ltd.; Sephadex LH-20 dextran gel from Shanghai Huicheng Biotechnology Co., Ltd.; pectinase with an activity of 30,000 / g from Qingdao Haiweisen Biotechnology Co., Ltd.; xylanase with an activity of 100,000 / g from Xi'an Qiannuo Bioengineering Co., Ltd.; bromelain with an activity of 100,000 / g from Qingdao Hezhan Biotechnology Co., Ltd.; and Bacillus subtilis protease with an activity of 20,000 / g from Jiangsu Duoyang Bioengineering Technology Co., Ltd. AB-8 macroporous adsorption resin was sourced from Tianjin Bohong Resin Technology Co., Ltd., pharmaceutical grade. Tanshinone IIA was sourced from Xi'an Muguo Biotechnology Co., Ltd., 98% purity. Panax notoginseng saponin R1 was sourced from Xi'an Jinhekang Pharmaceutical Co., Ltd., 90% purity. Ligustrazine was sourced from Shaanxi Jinkangtai Biotechnology Co., Ltd., 98% purity. Rhodiola rosea glycosides were sourced from Peptia Biotechnology Co., Ltd., 98% purity. Total flavonoids from hawthorn leaves were sourced from Xi'an Shennong Biotechnology Co., Ltd., 80% purity. Total flavonoids from mulberry leaves were sourced from Xi'an Hongshengkang Biomedical Technology Co., Ltd., 50% purity.

[0056] Comparative Example 1

[0057] The difference from Example 1 is that the fermented soybeans were changed to 45 parts and the extract of dried mushroom was changed to 8 parts.

[0058] Comparative Example 2

[0059] The difference from Example 1 is that the fermented soybeans were changed to 45 parts and the Akebia quinata peel extract was changed to 6 parts.

[0060] Comparative Example 3

[0061] The difference from Example 1 is that the extract of *Gnaphalium affine* was changed to 8 parts and the extract of *Akebia quinata* peel was changed to 26 parts.

[0062] Comparative Example 4

[0063] The difference from Example 1 is that the total flavonoids of hawthorn leaves are changed to 4.5 parts and the total flavonoids of mulberry leaves are changed to 0.5 parts.

[0064] Comparative Example 5

[0065] The difference from Example 1 is that the total flavonoids of hawthorn leaves are changed to 1 part and the total flavonoids of mulberry leaves are changed to 4 parts.

[0066] Comparative Example 6

[0067] The difference from Example 1 is that Bacillus coagulans is not used for the second stage of fermentation in the preparation of fermented soybeans.

[0068] Comparative Example 7

[0069] The difference from Example 1 is that fig protease is not used for enzymatic hydrolysis in the preparation of the Ganba mushroom extract.

[0070] Comparative Example 8

[0071] The difference from Example 1 is that in the preparation of the Ganba fungus extract, fig protease was replaced with papain (enzyme activity of 100,000 / g).

[0072] Comparative Example 9

[0073] The difference from Example 1 is that in the preparation of the *Gynostemma pentaphyllum* extract, the Sephadex LH-20 dextran gel column was replaced with a Sephadex LH-60 dextran gel column (from the same manufacturer).

[0074] Comparative Example 10

[0075] The difference from Example 1 is that the preparation of the akebia quinata peel extract does not use Bacillus subtilis protease for enzymatic hydrolysis.

[0076] Comparative Example 11

[0077] The difference from Example 1 is that in the preparation of the akebia peel extract, bromelain was replaced by papain (enzyme activity of 100,000 / g).

[0078] Comparative Example 12

[0079] The difference from Example 1 is that in the preparation of the Akebia quinata peel extract, the Bacillus subtilis protease was replaced by papain (enzyme activity of 100,000 / g).

[0080] Comparative Example 13

[0081] The difference from Example 1 is that in the preparation of the akebia quinata peel extract, the AB-8 macroporous adsorption resin column was replaced with D101 macroporous adsorption resin (from the same manufacturer).

[0082] I. Acute toxicity test (limit test)

[0083] 1. Experimental objective:

[0084] A preliminary assessment of the drug's acute toxicity was conducted to determine whether any significant acute toxic reactions were present.

[0085] 2. Experimental materials:

[0086] Animals: Healthy adult Kunming mice (18-22g, half male and half female), SPF grade, acclimatized to the environment for 3 days, excluding individuals with abnormal appearance / behavior.

[0087] Drugs and controls: Drug powders for each example and comparative example; blank control was 0.9% physiological saline (consistent with the drug solvent).

[0088] 3. Experimental design and drug administration:

[0089] Grouping: blank control group and drug group (each example and comparative example), 6 animals in each group (3 males and 3 females), with a weight difference of ≤10% between groups.

[0090] Dosage: Set at 2000 mg / kg body weight, the drug is prepared into a 100 mg / mL suspension with normal saline, and the gavage volume is 0.2 mL / 20 g; the blank group is given an equal volume of normal saline.

[0091] Method: Mice were fasted for 12 hours (with no restriction on water) and then administered the drug via gavage in a single dose. After administration, they were allowed to eat freely again.

[0092] 4. Observation and Recording:

[0093] Cycle: Observe for 4 hours immediately after administration, then twice daily (9 am and 5 pm) for a total of 7 days.

[0094] Indicators: Symptoms of poisoning (activity, hair, respiration, digestion, neurological response and death); weighing before administration and on days 1, 3, 7 and 14.

[0095] 5. Experimental Results:

[0096] No mice died or showed signs of poisoning in any group, and their body weight increased normally (P>0.05 between groups). The acute toxicity evaluation was negative.

[0097] II. Long-term toxicity test (120-day repeated administration)

[0098] 1. Experimental objective:

[0099] Assess the potential toxicity of long-term repeated administration, and clarify target organ toxicity and the scope of safe use.

[0100] 2. Experimental materials:

[0101] Animals: Same as acute toxicity test, acclimatization for 3 days, excluding underlying diseases.

[0102] Drugs and controls: Drug powders for each example and comparative example; blank control was 0.5% CMC-Na solution.

[0103] 3. Experimental design and drug administration:

[0104] Grouping: blank control group and drug group, 6 animals in each group (3 males and 3 females), with balanced weight and sex among groups.

[0105] Dosage: Set at 1 mg / 20 g body weight. Prepare a 50 mg / mL suspension with 0.5% CMC-Na and administer via gavage at a volume of 0.2 mL / 20 g.

[0106] Method: Gavage was administered at 9:00 AM daily for 120 consecutive days; the control group was given an equal volume of 0.5% CMC-Na.

[0107] 4. Observation and Testing:

[0108] During the period, general health and activity levels were observed, blood biochemistry was performed 24 hours after the last dose, and organ pathology was examined.

[0109] 5. Experimental Results:

[0110] No mice died or showed abnormalities in any group. There were no differences in body weight and food intake among the groups (P>0.05). Blood biochemical indicators were all within the normal range and showed no difference from the control group (P>0.05). Organ coefficients were normal and there were no drug-related pathological changes.

[0111] III. In vitro antiplatelet aggregation assay (turbidimetric method)

[0112] 1. Experimental objective:

[0113] To evaluate the ability of drugs to inhibit adenosine diphosphate (ADP)-induced platelet aggregation, reflecting their antithrombotic effects.

[0114] 2. Experimental materials:

[0115] Test drug: Powdered pharmaceutical compositions of each example and comparative example.

[0116] Reagents and equipment: physiological saline, ADP inducer (500 μmol / L stock solution, stored at -20℃), 3.8% sodium citrate solution, sodium pentobarbital, healthy New Zealand white rabbits (2.5-3.0 kg), platelet aggregator (LBY-NJ4 type), centrifuge, 0.22 μm microporous filter membrane.

[0117] 3. Experimental steps:

[0118] Sample preparation: Accurately weigh the drug powder, prepare a 10 mg / mL stock solution with physiological saline, filter it through a 0.22 μm microporous membrane for sterilization; dilute to a final concentration of 100 μg / mL before use.

[0119] Preparation of platelet-rich plasma (PRP) and platelet-poor plasma (PPP):

[0120] Rabbits were anesthetized by intravenous injection of sodium pentobarbital (30 mg / kg) into the ear margin vein, and blood was drawn by carotid artery catheterization and anticoagulated with 3.8% sodium citrate at a ratio of 9:1.

[0121] Anticoagulated blood was centrifuged at 800 rpm for 10 minutes, and the supernatant was collected as PRP; the remaining blood was centrifuged at 3000 rpm for 20 minutes, and the supernatant was collected as PPP.

[0122] Use PPP to adjust the PRP platelet count to 5×10. 8 per mL.

[0123] Testing procedures:

[0124] Preheat the platelet aggregation analyzer to 37°C and set the detection time to 6 minutes.

[0125] Add the sample to the turbidimetric tube according to the following formula:

[0126] Blank tube: 270 μL PRP + 30 μL physiological saline.

[0127] Positive control tube: 270 μL PRP + 30 μL aspirin solution (final concentration 100 μg / mL).

[0128] Dosing tube: 270 μL PRP + 30 μL test solution (final concentration 100 μg / mL).

[0129] After incubating the turbidimetric tube in the preheated well for 5 minutes, add 10 μL of ADP solution (final concentration 5 μmol / L), start aggregation recording, and the instrument will automatically generate a transmittance change curve.

[0130] Data calculation: Using the maximum aggregation rate (MAR) as the core indicator, the inhibition rate of the drug on platelet aggregation was calculated.

[0131] Inhibition rate (%) = [(MAR of blank group - MAR of treated group) / MAR of blank group] × 100%

[0132] Each group was tested three times (n=3), and the results are shown in Table 1 below.

[0133] IV. In vitro thrombolysis experiment (fibrin plate method)

[0134] 1. Experimental objective:

[0135] Qualitative and quantitative evaluations are conducted to assess the ability of drugs to dissolve fibrin, reflecting their thrombolytic activity.

[0136] 2. Experimental materials:

[0137] Test drug: Powdered pharmaceutical compositions of each example and comparative example.

[0138] Reagents and equipment: 0.05 mol / L Tris-HCl buffer (pH 7.4), fibrinogen (95% purity), thrombin (50 U / mL), urokinase (1000 U / mL, positive control), physiological saline, 6 cm culture dish, sterile punch (3 mm diameter), vernier calipers, 37℃ incubator.

[0139] 3. Experimental steps:

[0140] Sample preparation: Same as in Experiment 3, dilute the drug stock solution to a final concentration of 2 mg / mL.

[0141] Preparation of fibrin plates:

[0142] Prepare a Tris-HCl buffer (pH 7.4) containing 2 mg / mL fibrinogen, and add 5 mL to a 6 cm culture dish.

[0143] Add 100 μL of thrombin solution quickly, shake gently, and incubate at 37°C for 30 minutes to form an opaque fibrin gel plate.

[0144] 4. Thrombolysis test:

[0145] Use a punch to make even holes on the flat surface (1cm spacing between holes), and carefully remove the gel from the holes.

[0146] Add 20 μL of sample to each well (blank group: physiological saline; positive control group: urokinase; drug group: drug solution), and incubate the plate in a humidified chamber at 37°C for 18 hours.

[0147] 5. Data recording: The diameter of each weld ring was measured with vernier calipers (accurate to 0.1 mm). Three parallel holes were set for each group (n=3). The results are shown in Table 1 below.

[0148] V. In vitro antioxidant activity assay (DPPH free radical scavenging method)

[0149] 1. Experimental objective:

[0150] The ability of a drug to scavenge DPPH free radicals was determined, and the half-maximal scavenging concentration (IC50) was calculated. 50 This reflects its antioxidant activity (to aid in the assessment of vascular protective effects).

[0151] 2. Experimental materials:

[0152] Test drug: Powdered pharmaceutical compositions of each example and comparative example.

[0153] Reagents and equipment: anhydrous ethanol, DPPH (1,1-diphenyl-2-trinitrophenylhydrazine, purity 98%), microplate reader (517nm wavelength), 96-well plate, ultrasonic cleaner, centrifuge.

[0154] 3. Experimental steps:

[0155] Sample preparation: Accurately weigh the drug powder, prepare a 1.0 mg / mL stock solution with anhydrous ethanol, sonicate to dissolve (200W, 15 minutes), centrifuge at 5000 rpm for 5 minutes, and take the supernatant; then dilute to a series of concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL.

[0156] Preparation of DPPH solution: Prepare a 0.2 mM DPPH solution with anhydrous ethanol and store it protected from light (prepare and use immediately).

[0157] Testing procedures:

[0158] Add the following formulation to a 96-well plate (total system 200 μL per well):

[0159] Sample set: 100 μL sample solution + 100 μL DPPH solution.

[0160] Sample background group: 100 μL sample solution + 100 μL anhydrous ethanol (to eliminate interference from the sample's own absorbance).

[0161] Blank control group: 100 μL anhydrous ethanol + 100 μL DPPH solution.

[0162] - Gently shake to mix, cover and protect from light, react at room temperature for 30 minutes; measure the absorbance (OD value) of each well using a microplate reader at a wavelength of 517 nm.

[0163] Data calculation:

[0164] Clearance rate (%) = [1 - (OD sample - OD sample background) / OD blank control] × 100%; plot the dose-response curve with drug concentration on the x-axis and clearance rate on the y-axis, and calculate IC50 by linear regression. 50 Values; three replicates were set for each concentration (n=3), and the results are shown in Table 1 below.

[0165] VI. Effects on blood rheology and coagulation function in rats with blood stasis model

[0166] 1. Experimental objective:

[0167] The in vivo evaluation of the drug's effect on improving "blood stasis syndrome" was conducted to verify its efficacy in promoting blood circulation and removing blood stasis.

[0168] 2. Experimental materials:

[0169] Experimental animals: SPF-grade male SD rats, 200-220g.

[0170] Test drug: powdered drug compositions of each example and comparative example; positive control drug: Compound Danshen Dripping Pills (270 mg / kg).

[0171] Reagents and equipment: 0.5% CMC-Na solution, epinephrine hydrochloride injection, sodium pentobarbital, heparin sodium anticoagulant, 3.8% sodium citrate solution, fully automated blood rheology analyzer, centrifuge.

[0172] 3. Experimental steps:

[0173] Grouping and Sample Preparation:

[0174] Rats were randomly divided into groups of 6: blank group, model group, positive drug group, Example 1 group, Example 2 group, Example 3 group, and comparative examples 1-13 groups.

[0175] Both the drug and the positive control were prepared into a 20 mg / mL suspension using 0.5% CMC-Na (dose 200 mg / kg, volume 10 mL / kg); the blank group and the model group were given the same volume of 0.5% CMC-Na.

[0176] Modeling and drug administration:

[0177] Prophylactic administration: Except for the control group, all other groups were administered the drug once daily by gavage for 7 consecutive days; the control group and the model group were administered an equal volume of solvent by gavage.

[0178] Preparation of acute blood stasis model: One hour after the last administration on day 7, except for the blank group, rats in the other groups were subcutaneously injected with adrenaline hydrochloride injection (0.4 mg / kg) twice (4 hours apart); between the two injections, the rats were immersed in ice water for 5 minutes.

[0179] The control group received an equal volume of physiological saline subcutaneously during the same period, without ice water stimulation.

[0180] Sample collection:

[0181] Two hours after the last injection of epinephrine, anesthesia was administered via intraperitoneal injection of sodium pentobarbital (40 mg / kg), and blood was drawn from the abdominal aorta.

[0182] 5mL blood + heparin sodium anticoagulation: used for blood rheology testing.

[0183] 3 mL blood + 3.8% sodium citrate (9:1): Centrifuge at 3000 rpm for 15 minutes, and use the PPP sample for coagulation function testing.

[0184] 4. Indicator Testing:

[0185] Blood rheology: Automated blood rheology analyzer detected whole blood viscosity (low shear 5s). -1 ), and erythrocyte aggregation index. The test results are shown in Table 2 below.

[0186] 5. Statistical analysis: Data are expressed as "x±s". One-way ANOVA was performed using SPSS software. LSD method was used for pairwise comparisons between groups. P<0.05 was considered statistically significant.

[0187] Table 1. Results of in vitro pharmacodynamic experiments (mean value, n=3)

[0188]

[0189]

[0190] Table 2. Effects of the effects on blood rheology and coagulation function in rats with blood stasis (mean value, n=6)

[0191]

[0192]

[0193] Data analysis of Examples 1 to 3:

[0194] The data from the three embodiments show significant advantages, indicating that their formulation ratios and preparation processes are the optimal combination, resulting in a powerful synergistic effect.

[0195] Synergistic Blood-Activating Mechanism: Tanshinone IIA (calcium channel blocker, antioxidant), Panax notoginseng saponin R1 (anticoagulant, protects vascular endothelium), and ligustrazine (vasodilator, antiplatelet) are the core blood-activating components. Various active peptides and amino acids produced by fermented soybeans, as well as multiple active peptides provided by extracts of *Akebia quinata* and *Akebia quinata* peel, synergistically enhance bioavailability and blood-activating effects. Furthermore, the polysaccharides and flavonoids abundant in *Akebia quinata* and *Akebia quinata* peel extracts stabilize vascular endothelial cells through powerful antioxidant and anti-inflammatory effects, fundamentally reducing platelet activation and adhesion, creating a better environment for the core blood-activating components to function.

[0196] Technological advantages: The staged fermentation and compound enzymatic hydrolysis process maximizes the extraction and transformation of the special active ingredients in natural raw materials, thereby improving efficacy.

[0197] Analysis of Comparative Examples 1 to 5 (with changes in formulation ratios):

[0198] Comparative Example 1: The unique polysaccharides (such as β-glucan), bioactive peptides, and sterols abundant in *Gynostemma pentaphyllum* extract have immunomodulatory and anti-inflammatory effects, which weaken the overall systemic regulatory capacity of the drug. Although fermented soybeans increased the amount, they could not completely replace the immunomodulatory function of *Gynostemma pentaphyllum* extract, resulting in a weakness in the synergistic network and a decrease in efficacy.

[0199] Comparative Example 2: Akebia quinata rind extract is an important source of flavonoids and pectin polysaccharides; its reduction directly leads to a decrease in the drug's antioxidant capacity and ability to reduce blood viscosity. The addition of fermented soybeans cannot compensate for this functional deficiency because the two have different active ingredients and mechanisms of action.

[0200] Comparative Example 3: This ratio disrupted the formula balance. The reduction of the *Akebia quinata* extract resulted in the loss of immunomodulatory components, while the excessive amount of *Akebia quinata* peel extract, due to its highly viscous polysaccharides, slightly increased plasma viscosity. Furthermore, its active ingredients had reached absorption saturation and failed to achieve the expected effect, proving that "more is better" is not always true.

[0201] Comparative Examples 4 and 5: Total flavonoids from hawthorn leaves (lipid-lowering and cardiotonic) and total flavonoids from mulberry leaves (antioxidant and anti-inflammatory) form a stable "antioxidant microenvironment" when in the optimal ratio. When the ratio is changed, this microenvironment is disrupted, its protective efficiency against vascular endothelium and its ability to combat LDL oxidation are weakened, indirectly causing the core blood-activating components to have to cope with a more severe pathological environment, thus reducing the overall efficacy.

[0202] Analysis of Comparative Examples 6 to 13 (with changes in preparation process):

[0203] Comparative Example 6: The fermentation stage of Bacillus coagulans is crucial. It not only produces lactic acid to improve flavor, but also generates various antimicrobial peptides and functional enzymes, further breaking down anti-nutritional factors and releasing bound minerals and bioactive peptides. Without this stage, the functional component profile of fermented soybeans becomes limited, and its role in enhancing blood circulation and nourishing blood vessels is significantly reduced.

[0204] Comparative Example 7: This is one of the most significant process changes. The cell wall of *Bacillus thuringiensis* contains chitin, making it extremely tough. Cellulase and chitinase can only break down the cell wall, while the protein network inside the fungus requires proteases (figase) for targeted hydrolysis to release functional peptides, amino acids, and protein-bound active substances. Without proteases, most of the active substances are "locked" inside the cell, resulting in a sharp decrease in extract yield and activity.

[0205] Comparative Example 8: Fig protease exhibits extremely high proteolytic activity and a wide range of action sites. Papain differs from fig protease in its substrate specificity (its preference for hydrolyzing certain peptide bonds). This difference leads to alterations in the peptide sequence, length, and biological activity of the hydrolysate, resulting in a final product with functional properties inferior to the original process.

[0206] Comparative Example 9: Sephadex LH-20 and Sephadex LH-60 differ in their molecular sieve range and adsorption performance. LH-20's separation range (100-4000 Da) is more suitable for purifying target products such as flavonoids, aglycones, and small peptides. LH-60's wider separation range (100-20000 Da) leads to the co-elution of target small-molecule active ingredients with some large-molecule impurities, changes in the elution curve, decreased final product purity, and the presence of ineffective or low-efficiency components, thus diluting the activity.

[0207] Comparative Example 10: Pectin and polysaccharides in the rind of the August melon often form complexes with proteins. The lack of Bacillus subtilis protease prevents these complexes from being fully broken down, leading to a reduced extraction rate of pectin polysaccharides and a lower efficiency in removing insoluble fiber. This results in the final extract containing more impurities and insufficient purity of the active ingredients. Furthermore, the structure of the enzymatically hydrolyzed protein peptides also changes, affecting the therapeutic effect.

[0208] Comparative Examples 11 and 12: The substitution of papain resulted in different enzymatic hydrolysis mechanisms and efficiencies, leading to different amino acid sequences in the resulting protein peptides. This resulted in differences in the direction and effect of action, ultimately affecting the product composition and synergistic effect of the drug. The complex could not be fully decomposed, leading to a decrease in the extraction rate of pectin polysaccharides and the removal efficiency of insoluble fibers. The final extract contained more impurities, resulting in insufficient purity of the active ingredients.

[0209] Comparative Example 13: AB-8 resin is a weakly polar resin, and its pore size and polarity are very suitable for adsorbing and separating moderately polar natural products, such as flavonoids, saponins, and phenolic acids. D101 resin is a non-polar resin with strong adsorption capacity for lipid-soluble components (such as chlorophyll and essential oils), but its adsorption and desorption rates for moderately polar components such as flavonoids are not as good as AB-8. Changing the resin altered the adsorption-elution kinetics, leading to changes in the retention behavior of the target components. Some components could not be eluted or were interfered with by impurities, ultimately affecting the purity and activity of the extract.

Claims

1. A blood-activating and stasis-removing pharmaceutical composition containing fermented soybeans, characterized in that, The pharmaceutical composition consists of the following raw materials in parts by weight: 30-40 parts fermented soybean, 15-20 parts dried mushroom extract, 15-20 parts Akebia quinata peel extract, 10-15 parts tanshinone IIA, 8-12 parts notoginseng saponin R1, 5-10 parts ligustrazine, 3-5 parts rhodioloside, 3-4 parts total flavonoids from hawthorn leaves, and 1-2 parts total flavonoids from mulberry leaves; The fermented soybean paste is made from soybeans and black beans through fermentation with Bacillus subtilis and Bacillus coagulans. The extract of *Gymnosus chinensis* was prepared by enzymatic hydrolysis of *Gymnosus chinensis* powder with cellulase, chitinase and β-glucanase, followed by biphasic solvent extraction and purification by Sephadex LH-20 glucan gel column chromatography. The August melon peel extract was prepared by enzymatic hydrolysis of August melon peel powder by pectinase, cellulase and xylanase, enzymatic hydrolysis by bromelain and subtilisin, and purification by AB-8 macroporous adsorption resin column.

2. The blood-activating and stasis-removing pharmaceutical composition containing fermented soybeans according to claim 1, characterized in that, The preparation of the fermented soybean paste includes: soybeans and black beans in a mass ratio of 1:(0.4-0.6) are steamed and sterilized, and then fermented sequentially by Bacillus subtilis and Bacillus coagulans to obtain the product; The preparation of the *Gnaphalium affine* extract includes: enzymatically hydrolyzing *Gnaphalium affine* powder with cellulase, chitinase, and β-glucanase to obtain enzymatically hydrolyzed bacterial powder, then extracting it with a two-phase solvent prepared with ethanol, water, and ammonium sulfate, taking the upper phase, passing it through a Sephadex LH-20 dextran gel column, and washing it successively with deionized water, 30%–35% (v / v) ethanol aqueous solution, and 50%–55% (v / v) ethanol aqueous solution to remove impurities, eluting it with 70%–75% (v / v) ethanol aqueous solution, collecting the eluent, concentrating it under reduced pressure, and freeze-drying it to obtain the product; The preparation of the August melon rind extract includes: washing August melon rind powder with sodium bicarbonate aqueous solution, followed by enzymatic hydrolysis with pectinase, cellulase and xylanase, then adding bromelain and subtilisin to obtain the enzymatic hydrolysate, centrifuging, collecting the supernatant, concentrating under reduced pressure, loading the sample onto an AB-8 macroporous adsorption resin column, first rinsing with deionized water to remove impurities, then eluting with 50% to 60% ethanol aqueous solution for 4 to 5 column volumes, collecting the eluent, concentrating under reduced pressure, and freeze-drying to obtain the product.

3. The blood-activating and stasis-removing pharmaceutical composition containing fermented soybeans according to claim 1, characterized in that, The preparation method of the fermented soybeans includes: steaming and sterilizing soybeans, cooling to 35℃~38℃, adding sterile water, inoculating with Bacillus subtilis activated bacterial solution under stirring, and carrying out aerobic fermentation at 35℃~38℃ for 20h~24h, heating up for sterilization, cooling to 40℃~45℃, inoculating with Bacillus coagulans activated bacterial solution under stirring, and carrying out aerobic fermentation at 40℃~45℃ for 16h~18h, heating up for sterilization, drying, and pulverizing into powder to obtain fermented soybeans.

4. The blood-activating and stasis-removing pharmaceutical composition containing fermented soybeans according to claim 3, characterized in that, The soybeans are yellow soybeans and black soybeans in a mass ratio of 1:(0.4-0.6); the soybeans are soaked and drained before steaming and sterilization; the steaming and sterilization is carried out at a temperature of 115℃-122℃ and a steam pressure of 0.12MPa-0.14MPa for 40-50 minutes; the amount of sterile water used is 30%-35% of the soybean mass; the inoculum amount of Bacillus subtilis activated bacterial solution is 4%-6% of the soybean mass; the concentration of the Bacillus subtilis activated bacterial solution is 1.0×10⁻⁶. 9 CFU / mL ~5.0×10 9 CFU / mL; the inoculation amount of the activated Bacillus coagulans solution is 2%–3% of the soybean mass; the concentration of the activated Bacillus coagulans solution is 5.0 × 10⁻⁶ CFU / mL. 8 CFU / mL ~ 2.0 × 10 9 CFU / mL; the temperature sterilization was carried out at 80℃~90℃ for 10min~20min; the drying was carried out at 50℃~60℃ until the moisture content was less than 5wt%.

5. The blood-activating and stasis-removing pharmaceutical composition containing fermented soybeans according to claim 1, characterized in that, The preparation method of the *Gynostemma pentaphyllum* extract includes: adding citrate-disodium hydrogen phosphate buffer to *Gynostemma pentaphyllum* powder, then adding cellulase, chitinase, and β-glucanase, stirring and hydrolyzing at 45℃~50℃ for 2h~3h, then adding fig protease, stirring and hydrolyzing at 60℃~65℃ for 1.5h~2h, inactivating the enzyme by heating, concentrating under reduced pressure, and freeze-drying to obtain the enzymatically hydrolyzed bacterial powder; preparing a biphasic solvent at a mass ratio of ethanol:water:ammonium sulfate = (25~30):(70~75):(1~1.5); adding the enzymatically hydrolyzed bacterial powder to the biphasic solvent at a material-to-liquid ratio of 1g:12mL~1g:15mL, and adding 0.1%~0.2% L-ascorbic acid by mass of the enzymatically hydrolyzed bacterial powder, sonicating, allowing to stand and separate into layers, taking the upper phase, and loading the sample onto Sephadex. The LH-20 dextran gel column was eluted sequentially with one column volume each of deionized water, 30%–35% (v / v) ethanol aqueous solution, and 50%–55% (v / v) ethanol aqueous solution to remove impurities. Finally, it was eluted with 70%–75% (v / v) ethanol aqueous solution for 2–3 column volumes. The eluent was collected, concentrated under reduced pressure to remove ethanol, and freeze-dried to obtain the *Bacillus thuringiensis* extract.

6. The blood-activating and stasis-removing pharmaceutical composition containing fermented soybeans according to claim 5, characterized in that, The *Gynostemma pentaphyllum* powder is obtained by drying *Gynostemma pentaphyllum* below 60℃, pulverizing it, and passing it through a 200-250 mesh sieve; the amount of citrate-disodium hydrogen phosphate buffer added is 6-8 times the mass of the *Gynostemma pentaphyllum* powder; the pH value of the citrate-disodium hydrogen phosphate buffer is 5.0-5.5; the amount of cellulase, chitinase, and β-glucanase added is 0.5%-1% of the mass of the *Gynostemma pentaphyllum* powder; the amount of fig protease added is 0.8%-1.5% of the mass of the *Gynostemma pentaphyllum* powder; the stirring speed for enzymatic hydrolysis is 50-80 rpm; the enzyme inactivation by heating to 85-90℃ for 10-15 minutes; the ultrasonic treatment is performed at 50-55℃ and 300-350W power for 30-60 minutes; the temperature for vacuum concentration is 50-60℃.

7. The blood-activating and stasis-removing pharmaceutical composition containing fermented soybeans according to claim 1, characterized in that, The preparation method of the August melon rind extract includes: adding August melon rind powder to a sodium bicarbonate aqueous solution, stirring, centrifuging, and collecting the precipitate; adding 8 to 12 times the mass of water to adjust the pH to 4.5 to 5.0, then adding pectinase, cellulase, and xylanase, and performing enzymatic hydrolysis for 2 to 3 hours at 50°C to 55°C and 50 to 80 rpm, adjusting the pH to 6.5 to 7.5, adding bromelain and subtilisin, and then performing enzymatic hydrolysis at 50°C to 55°C. The mixture was stirred at 50-80 rpm for 1-2 hours to undergo a second enzymatic hydrolysis at ℃. The enzyme was then inactivated by heating and cooled to 60-65℃ to obtain the hydrolysate. The hydrolysate was cooled, centrifuged, and the supernatant was collected. The supernatant was concentrated under reduced pressure and loaded onto an AB-8 macroporous adsorption resin column. The column was first rinsed with 2-3 column volumes of deionized water to remove impurities, and then eluted with 50-60% ethanol aqueous solution for 4-5 column volumes. The eluent was collected, concentrated under reduced pressure to remove ethanol, and then freeze-dried to obtain the akebia quinata peel extract.

8. The blood-activating and stasis-removing pharmaceutical composition containing fermented soybeans according to claim 7, characterized in that, The August melon rind powder is obtained by taking the rinds of August melons that are 80% ripe or more, drying them below 60℃, pulverizing them, and passing them through a 200-250 mesh sieve; the amount of sodium bicarbonate aqueous solution used is 4 to 6 times the mass of the August melon rind powder; the concentration of the sodium bicarbonate aqueous solution is 0.5wt% to 0.8wt%; the stirring is carried out at 40℃ to 45℃ and 50rpm to 80rpm for 30 to 60 minutes; the amount of pectinase, cellulase, and xylanase added is 0.5% to 1.5% of the mass of the August melon rind powder; the amount of bromelain and subtilisin added is 0.5% to 1.5% of the mass of the August melon rind powder; the enzyme inactivation is carried out by heating to 85℃ to 90℃ for 10 to 15 minutes; the centrifugation is carried out at a speed of 6000rpm to 8000rpm for 10 to 15 minutes; the temperature for vacuum concentration is 50℃ to 60℃.

9. A method for preparing a blood-activating and stasis-removing pharmaceutical composition containing fermented soybeans as described in any one of claims 1 to 7, characterized in that, The process includes the following steps: mixing the formulated amounts of fermented soybeans, *Gnaphalium affine* extract, *Akebia quinata* peel extract, tanshinone IIA, notoginsenoside R1, ligustrazine, rhodioloside, total flavonoids from hawthorn leaves, and total flavonoids from mulberry leaves evenly to obtain a pharmaceutical composition; the pharmaceutical composition is then mixed with pharmaceutically acceptable excipients to prepare any pharmaceutically acceptable dosage form, including tablets, capsules, granules, pills, powders, and oral liquids.

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