A brain-nourishing and meridian-unblocking traditional Chinese medicine granule and a preparation method thereof

Abstract

The application relates to the technical field of traditional Chinese medicines, and particularly discloses a brain-benefiting and meridian-unblocking traditional Chinese medicine granule and a preparation method thereof, which comprises traditional Chinese medicine active raw medicine and medicinal excipients; the active raw medicine is composed of the following components in proportion by weight: motherwort 20-25 parts, prepared radix falcaliae 12-17 parts, radix puerariae 12-17 parts, chuanxiong rhizome 12-17 parts, eucommia ulmoides 12-17 parts, safflower 9-13 parts and alisma 5-9 parts; the granule has a triple linkage brain protection mechanism in improving hemodynamics, resisting oxidative stress and targeting inhibiting ferroptosis, provides systematic data support for the preclinical effectiveness of the product in preventing and treating ischemic stroke, is different from a traditional decoction rough medication mode, and provides a definite drug efficacy for the clinical prevention and treatment of ischemic stroke.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a brain-boosting and meridian-clearing traditional Chinese medicine granule and its preparation method. Background Technology

[0002] Stroke, especially ischemic stroke, is one of the leading causes of death and disability worldwide, characterized by high incidence, high disability rate, high recurrence rate, and high mortality rate, imposing a heavy disease burden on society and patients' families. Cerebral ischemia-reperfusion injury is an unavoidable key pathological step in the clinical treatment of ischemic stroke, triggering a series of complex cascade reactions such as oxidative stress, inflammatory response, and programmed cell death, further aggravating brain tissue damage. It remains a core challenge in the field of stroke treatment.

[0003] Current clinical treatments for ischemic stroke remain significantly limited. In modern medicine, ultra-early intravenous thrombolysis is the primary method for vascular recanalization, with alteplase injection as a representative drug. However, its therapeutic window is only within 4.5 hours of onset, and it carries a serious risk of intracranial hemorrhage as a side effect. Only a very small number of patients can receive timely and standardized thrombolytic therapy. Edaravone and other free radical scavenging neuroprotective agents have limitations in their single target and overall benefit, and their clinical application value remains controversial. Overall, modern medicine still lacks effective treatment options that can block the pathological process of cerebral ischemia-reperfusion injury at multiple stages and targeting points.

[0004] Traditional Chinese medicine has accumulated thousands of years of clinical experience in the prevention and treatment of stroke. The experience prescriptions of famous veteran Chinese medicine doctors, as a concentrated embodiment of the clinical wisdom of Chinese medicine, have unique advantages of overall regulation and multi-target intervention, and are an important source of innovative research and development of Chinese medicine.

[0005] However, most of the experienced prescriptions of renowned traditional Chinese medicine practitioners are still used in clinical practice in the form of traditional decoctions, which have many inherent defects: First, the decoction preparation process is cumbersome, inconvenient to carry and take, and patients have poor long-term medication compliance; second, the manual decoction extraction method is crude, and the dissolution rate of effective ingredients fluctuates greatly due to the influence of decoction conditions, resulting in insufficient stability of clinical efficacy; third, there is a lack of standardized preparation processes and quality control systems, resulting in significant batch-to-batch quality differences, making it difficult to achieve industrialized and large-scale production, which seriously restricts the clinical promotion and modernization of experienced prescriptions.

[0006] Among the marketed oral traditional Chinese medicines, many products used for stroke prevention and treatment focus on single effects such as promoting blood circulation and removing blood stasis. They lack systematic and integrated research on the complex pathological network involving hemodynamic disturbances, oxidative stress imbalances, and ferroptosis activation in cerebral ischemia-reperfusion injury. In recent years, ferroptosis, as a novel form of programmed cell death, has been proven to be an important pathological mechanism of cerebral ischemia-reperfusion injury. Drug development targeting the ferroptosis pathway has become a research hotspot in neuroprotection. However, to date, there are no publicly available technical solutions for developing granules of traditional Chinese medicine formulas targeting this pathway and for verifying their neuroprotective mechanism in conjunction with the ferroptosis pathway system. Summary of the Invention

[0007] The purpose of this invention is to provide a brain-nourishing and meridian-clearing traditional Chinese medicine granule and its preparation method, so as to solve the problem of insufficient research on existing traditional Chinese medicine preparations targeting the core frontier mechanisms of cerebral ischemia-reperfusion injury mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A brain-boosting and meridian-clearing traditional Chinese medicine granule includes active raw materials of traditional Chinese medicine and pharmaceutical excipients; the active raw materials are composed of the following components by weight: 20-25 parts of Leonurus japonicus, 12-17 parts of processed Polygonum multiflorum, 12-17 parts of Pueraria lobata, 12-17 parts of Ligusticum chuanxiong, 12-17 parts of Eucommia ulmoides, 9-13 parts of Carthamus tinctorius, and 5-9 parts of Alisma plantago-aquatica.

[0010] Preferably, the active pharmaceutical ingredient is composed of the following components by weight: 22.2 parts of Leonurus japonicus, 14.8 parts of processed Polygonum multiflorum, 14.8 parts of Pueraria lobata, 14.8 parts of Ligusticum chuanxiong, 14.8 parts of Eucommia ulmoides, 11.1 parts of Carthamus tinctorius, and 7.4 parts of Alisma plantago-aquatica.

[0011] Preferably, the pharmaceutical excipient is soluble starch; the mass ratio of the dry extract powder of the active pharmaceutical ingredient to the soluble starch is 1:1.

[0012] A method for preparing traditional Chinese medicine granules includes the following steps:

[0013] S1. Raw material pretreatment: Weigh each raw material according to the prescription amount, clean and select the processed Polygonum multiflorum, Eucommia ulmoides, and Alisma plantago-aquatica, crush them into 10-mesh coarse powder, and cut the remaining medicinal materials into slices; soak them in water until they are saturated with water.

[0014] S2. Water decoction extraction: The soaked medicinal materials are heated and refluxed twice for extraction, and the extracts are combined and filtered to remove residue;

[0015] S3. Vacuum Concentration: The extract is concentrated under reduced pressure to a clear paste with a relative density of 1.25 to 1.35 as measured by thermal analysis at 80°C;

[0016] S4. Drying and powdering: Dry the paste under reduced pressure to constant weight, pulverize and sieve to obtain dry paste powder;

[0017] S5. Wet granulation: Mix the dry powder with soluble starch evenly, and prepare wet granules using ethanol as a wetting agent;

[0018] S6. Drying and granulation: After drying the wet granules, granulate, mix, and package them to obtain the final product.

[0019] Preferably, in S1, add 6 to 10 times the amount of water and soak for 50 minutes.

[0020] Preferably, in S2, the first extraction is performed with 8 times the amount of water for 1.5 hours, and the second extraction is performed with 6 times the amount of water for 1.0 hour; the extract is filtered through a 200-mesh sieve to remove residue.

[0021] Preferably, in step S3, the temperature for vacuum concentration is 80℃ and the vacuum degree is -0.08MPa; in step S4, the temperature for vacuum drying is 70℃ and the vacuum degree is -0.08MPa, and the dried product is pulverized through a 100-mesh sieve.

[0022] Preferably, in step S5, the dry powder and soluble starch are added in equal amounts at a mass ratio of 1:1 and mixed evenly; the wetting agent is 75% ethanol by volume, and the amount used is 12% of the total mass of the powder; granulation is carried out using a 16-mesh sieve; in step S6, the drying temperature of the wet granules is 60℃ and the drying time is 3 hours; after drying, the granules are sieved through a 10-mesh sieve and an 80-mesh sieve, mixed for 15-30 minutes, and sealed and packaged in 20g bags.

[0023] This invention relates to the application of brain-nourishing and meridian-clearing traditional Chinese medicine granules in the preparation of drugs for the prevention and treatment of ischemic stroke.

[0024] Preferably, the drug is used to improve hemodynamics in ischemic brain regions, reduce oxidative stress damage in brain tissue, or inhibit ferroptosis in brain nerve cells. The drug activates the NRF2 signaling pathway, upregulates the expression of GPX4 and SLC7A11 proteins, and blocks the ferroptosis pathway induced by cerebral ischemia-reperfusion injury.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This invention addresses the technical challenges of the compound extract, including high hygroscopicity, poor flowability, and difficulty in granulation. Through excipient screening and process parameter optimization, a granulation scheme was determined using soluble starch as the optimal excipient, a drug-to-excipient mass ratio of 1:1, and 75% ethanol as a wetting agent (12%), achieving a granulation rate of 92.30%. Simultaneously, the entire preparation process was optimized, with the RSD of effective ingredient content in each stage of extraction, concentration, and drying all below 1.80%, demonstrating excellent process reproducibility. The finished granules fully meet the requirements for granules in the Chinese Pharmacopoeia, including particle size, moisture content, solubility, and fill weight variation. This solves the problems of cumbersome decoction preparation, large quality fluctuations, and poor patient compliance associated with traditional decoctions, and can be directly scaled up for large-scale industrial production.

[0027] Simultaneously, this invention provides multi-target synergistic neuroprotection, and for the first time clearly elucidates the regulatory mechanism of ferroptosis, systematically verifying the triple-linked neuroprotective efficacy: it can significantly improve blood rheological characteristics, exert a clear anticoagulant effect; reduce cerebral edema and pathological damage to brain tissue, improve neurological function, and reduce the volume of cerebral infarction; regulate oxidative stress and inflammatory response, and correct the redox imbalance in brain tissue. Furthermore, it is the first time that this preparation has been demonstrated to activate the NRF2 pathway, upregulate the expression of GPX4 and SLC7A11 proteins, and target and inhibit neuronal ferroptosis induced by cerebral ischemia-reperfusion injury, elucidating its scientific implications from a cutting-edge pathological perspective, distinguishing it from existing single-function blood-activating and stasis-removing traditional Chinese medicine preparations.

[0028] Meanwhile, this invention has clear anticoagulant and hemorheological improvement effects. Yinao Tongluo granules can significantly prolong coagulation time, regulate intrinsic / extrinsic coagulation pathways, and reduce fibrinogen in normal animals. Yinao Tongluo granules can also effectively reduce cerebral edema. By reducing blood viscosity, improving erythrocyte deformability and aggregation, prolonging coagulation time, and reducing fibrinogen, it can improve hemorheology and anticoagulation effects. At the same time, it can improve neurological function and brain tissue pathological damage after cerebral ischemia, regulate oxidative stress and inflammatory response, improve the redox imbalance in brain tissue, and upregulate the expression of key proteins in ferroptosis by activating the NRF2 pathway, thereby targeting and inhibiting ferroptosis.

[0029] In summary, the triple-linked neuroprotective mechanism of the granules of this invention in improving hemodynamics, resisting oxidative stress, and targeting and inhibiting ferroptosis provides systematic data support for the preclinical efficacy of this product in preventing ischemic stroke and clarifies its scientific significance in distinguishing it from the traditional crude method of decoction administration. Attached Figure Description

[0030] Figure 1 HE staining results of rats in each group 24 h after ischemia-reperfusion;

[0031] Figure 2 Nissl staining results of rats in each group 24 h after ischemia-reperfusion.

[0032] Figure 3 Western blot results of NRF2, HO-1, SLC7A11, and GPX4 proteins in rat brain tissue of each group. Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1: Preparation of Brain-Nourishing and Meridian-Clearing Granules

[0035] Prescription of this invention

[0036] Motherwort 222 Processed Polygonum multiflorum 148 Kudzu root 148 Chuanxiong 148 Eucommia 148 safflower 111 Alisma plantago-aquatica 74 total 999

[0037] Preparation method

[0038] (1) Raw material pretreatment and soaking: Weigh each raw material according to the prescription and clean it. Process Polygonum multiflorum, Eucommia ulmoides and Alisma plantago-aquatica into coarse powder through a 10-mesh sieve, and cut the rest into slices. Soak in 8 times the amount of water for 50 minutes.

[0039] (2) Water decoction extraction: Add 8 times the amount of water for the first extraction, heat and reflux for 1.5 hours, keeping it at a gentle boil; add 6 times the amount of water for the second extraction, extract for 1.0 hour, keeping it at a gentle boil. Combine the two extracts.

[0040] (3) Solid-liquid separation: Combine the extracts and filter them through a 200-mesh sieve to remove residue.

[0041] (4) Concentration under reduced pressure: The filtrate was concentrated under reduced pressure at 80℃ and -0.08MPa to a relative density of 1.25 to 1.35 (measured at 80℃).

[0042] (5) Vacuum drying: The concentrate was dried under reduced pressure at 70℃ and -0.08MPa until constant weight (moisture content ≤5.0%).

[0043] (6) Crushing and sieving: The dry extract is crushed and sieved through a 100-mesh sieve to obtain dry extract powder.

[0044] (7) Mixing of excipients: Take dry powder and soluble starch and mix them at a mass ratio of 1:1. Mix them evenly by adding them in equal amounts.

[0045] (8) Wet granulation: 75% ethanol, which accounts for 12% of the total amount of pharmaceutical excipients, is used as a wetting agent and granulation is performed using a 16-mesh sieve.

[0046] (9) Particle drying: Dry the wet particles at 60°C for 3 hours.

[0047] (10) Granulation, mixing and packaging: The dried granules are granulated by passing them through a No. 1 sieve (10 mesh) and a No. 5 sieve (80 mesh), and mixed for 15-30 minutes. Each bag is divided into 20g portions and packaged in high-density polyethylene plastic bottles, sealed and protected from moisture.

[0048] 3. Preparation Results

[0049] In this embodiment, three batches of granules were prepared (batch numbers 20201001, 20201002, and 20201003). The granules were brownish-red in appearance, with a slightly fragrant odor and a sweet and slightly bitter taste. The test results of various indicators are shown in Table 1.

[0050] Table 1. Routine inspection results of three batches of granules

[0051] 20201001 9.2 3.6 Compliant Compliant 20201002 8.5 3.1 Compliant Compliant 20201003 10.5 4.1 Compliant Compliant Pharmacopoeia stipulates ≤15 ≤5.0 Dissolve completely in ≤3 minutes Compliant

[0052] Example 2: Optimization Experiment of Extraction Process Parameters

[0053] 1. Orthogonal experimental design

[0054] Using water volume, extraction time, and extraction times as factors of investigation, and puerarin content and extract yield as evaluation indicators, the L9(3) model was adopted. 4 An orthogonal array was used for the experiment. The factor levels are shown in Table 2.

[0055] Table 2. Factor Level Table for Orthogonal Experiment

[0056] 1 June 4 1.0 / 0.5 1 2 August 6 1.5 / 1.0 2 3 October 8 2.0 / 1.5 3

[0057] 2. Results of the orthogonal experiment

[0058] Orthogonal experimental results showed that the influence of each factor on the puerarin content and extract yield was in the order of C (number of extractions) > A (amount of water added) > B (extraction time). The optimal extraction process was A2B2C2, that is, the first extraction was carried out with 8 times the amount of water for 1.5 hours, and the second extraction was carried out with 6 times the amount of water for 1.0 hour, for a total of 2 extractions.

[0059] 3. Verification Experiment

[0060] Three batches of verification experiments were conducted according to the optimized process, and the results are shown in Table 3.

[0061] Table 3 Results of the extraction process verification experiment

[0062] 1 31.52 74.89 68.52 2 30.98 74.12 69.31 3 31.38 74.74 68.78 mean 31.29 74.58 68.87 RSD (%) 1.06 0.63 0.67

[0063] The verification results show that the optimized process is stable, feasible, and has good reproducibility.

[0064] Example 3: Screening of Formulation Process Parameters

[0065] 1. Selection of excipient types

[0066] The dry extract powder was mixed with soluble starch, dextrin, lactose, mannitol, microcrystalline cellulose, and glucose in a 1:1 ratio, and granulated using 75% ethanol as a wetting agent. The ease of granulation and the appearance of the granules were examined. The results are shown in Table 4.

[0067] Table 4 Comparison of Granulation Effects of Different Excipients

[0068] Soluble starch easy Uniform particle size and consistent color dextrin relatively easy The particles are uniform and slightly sticky. lactose Difficult The particles are loose and contain a lot of fine powder. Mannitol Difficult Hard particles, uneven color microcrystalline cellulose generally Uniform particles and good flowability glucose Difficult Particles stick together and are difficult to dry

[0069] Based on comprehensive comparison, soluble starch was determined to be the best excipient.

[0070] 2. Screening of excipient dosage

[0071] Dry powder and soluble starch were mixed in different proportions and granulated using 75% ethanol as a wetting agent. The granulation rate, moisture content, and solubility were investigated. The results are shown in Table 5.

[0072] Table 5 Comparison of Granulation Effects with Different Amounts of Excipients

[0073] 01:00.5 86.79 4.1 clarify 1:01 90.83 4.55 clarify 01:01.5 89.86 4.87 clarify 1:02 88.12 5.12 A small amount of sediment was found. 01:02.5 88.65 5.36 A small amount of sediment was found.

[0074] Based on comprehensive comparison, the highest molding rate was determined when the ratio of dry powder to soluble starch was 1:1.

[0075] 3. Screening of wetting agent concentration

[0076] The dry extract powder and soluble starch were mixed in a 1:1 ratio, and the molding rate was examined using ethanol of different concentrations as a wetting agent (12% of the total amount of pharmaceutical excipients). The results are shown in Table 6.

[0077] Table 6 Comparison of granulation effects of different ethanol concentrations

[0078] 60 58.7 Loose particles, mostly fine powder 65 72.4 The particles are relatively loose 70 85.6 Uniform particle size 75 92.5 Uniform particle size and consistent color 80 88.3 The granules are slightly hard 85 82.1 The particles are hard and dark in color. 90 78.6 Hard particles, uneven color

[0079] 75% ethanol was determined to be the optimal wetting agent concentration.

[0080] 4. Screening of wetting agent dosage

[0081] The dry powder and soluble starch were mixed in a 1:1 ratio, and 75% ethanol was used as a wetting agent. The molding rate under different dosages was investigated. The results are shown in Table 7.

[0082] Table 7 Comparison of Granulation Effects with Different Wetting Agent Doses

[0083] 6 74.5 Loose particles, mostly fine powder 8 82.3 The particles are relatively loose 10 88.7 Uniform particle size 12 92.3 Uniform particle size and consistent color 13 90.1 The particles are slightly sticky 15 86.8 Severe particle adhesion

[0084] The highest molding rate was achieved when the wetting agent dosage was 12%.

[0085] 5. Drying temperature screening

[0086] The wet granules were dried at 50℃, 60℃, and 70℃ respectively, and the content of stachydrine hydrochloride and moisture were investigated. The results are shown in Table 8.

[0087] Table 8. Effects of different drying temperatures on the indicators

[0088] 50 0.41 4.52 60 0.45 3.96 70 0.38 2.85

[0089] The optimal drying temperature was determined to be 60℃.

[0090] Example 4: Thin-layer chromatography identification

[0091] 1. Identification of Ligusticum chuanxiong

[0092] Take 5g of the granules, add 20ml of ethyl acetate, sonicate for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of ethyl acetate to prepare the test solution. Separately, prepare a reference solution of ferulic acid by dissolving it in ethyl acetate to a concentration of 1mg / ml. Perform thin-layer chromatography (General Chapter 0502 of the Chinese Pharmacopoeia). Apply 5μl of each of the two solutions to the same silica gel GF film. 254 On a thin-layer plate, cyclohexane-ethyl acetate-formic acid (20:10:1) was used as the developing solvent. The plate was then removed, dried, and examined under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample. Spraying with a 1% ferric chloride-1% potassium ferricyanide (1:1) mixed solution for color development deepened the color of the spots. The negative control showed no interference.

[0093] 2. Identification of Kudzu Root

[0094] Take 5g of the granules of this product, add 20ml of 30% ethanol, sonicate for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of 30% ethanol, and use this as the test solution. Separately, take puerarin reference standard and pueraria lobata reference material, and prepare solutions containing 1mg / ml and 5mg / ml respectively with 30% ethanol, as the reference solution and reference material solution. Perform thin-layer chromatography, applying 5μl of each of the above three solutions to the same silica gel G thin-layer plate, using chloroform-methanol-water (7:2.5:0.25) as the developing solvent. Develop, remove, air dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent streaks of the same color appear at the corresponding positions as in the chromatograms of the reference standard and reference material. No interference is observed in the negative control.

[0095] 3. Identification of Safflower

[0096] Take 5g of the granules of this product, add 20ml of 80% acetone, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of 80% acetone, and use this as the test solution. Separately, take 1g of safflower reference material and prepare a reference material solution using the same method. Perform thin-layer chromatography, applying 5μl of each of the above two solutions separately to the same silica gel G thin-layer plate. Develop using dichloromethane-ethyl acetate-glacial acetic acid (5:4:1) as the developing solvent, remove, air dry, and examine under ultraviolet light (254nm). In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference material. There is no interference from the negative control.

[0097] Example 5: Determination of Puerarin Content

[0098] 1. Chromatographic conditions

[0099] Chromatographic column: Octadecylsilane bonded silica column (4.6 mm × 150 mm, 5 μm);

[0100] Mobile phase: methanol-water (25:75); detection wavelength: 250 nm; flow rate: 1.0 ml / min; column temperature: 30 ℃; injection volume: 10 μl.

[0101] The theoretical plate number, calculated based on the puerarin peak, should be no less than 3000.

[0102] 2. Preparation of reference solution

[0103] Accurately weigh an appropriate amount of puerarin reference standard, add 30% ethanol to prepare a solution containing 0.1 mg per ml.

[0104] 3. Preparation of the test solution

[0105] Take about 2g of the fine powder of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 30% ethanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, make up the weight loss with 30% ethanol, shake well, filter it, and take the filtrate to obtain the product.

[0106] 4. Examination of linear relationships

[0107] Accurately pipette 2, 4, 6, 8, and 10 μl of puerarin reference solution and inject them into the liquid chromatograph to determine the peak area. Plot a standard curve with peak area as the ordinate (Y) and injection volume (μg) as the abscissa (X), yielding the regression equation: Y = 92.709X - 3.9977, r 2 = 0.9992. The results indicate that puerarin exhibits good linearity in the range of 0.48 μg to 1.28 μg.

[0108] 5. Precision test

[0109] Accurately pipette 10 μl of the same reference solution and inject it 6 times consecutively. Measure the peak area and calculate the RSD to be 0.31%, indicating good instrument precision.

[0110] 6. Repeatability test

[0111] Six samples from the same batch (batch number 20201001) were taken, and test solutions were prepared and the contents were determined. The RSD was calculated to be 1.94%, indicating that the method has good repeatability.

[0112] 7. Stability Test

[0113] The same test solution was tested at 0, 2, 4, 6, 8, 12 and 24 h. The calculated RSD was 3.41%, indicating that the test solution had good stability within 24 h.

[0114] 8. Recovery test

[0115] Nine samples (batch number 20201001) with known content were taken, each approximately 1 g, and accurately weighed. Appropriate amounts of puerarin reference standard (equivalent to 80%, 100%, and 120% of the sample content) were added to each sample. The solutions were prepared and determined according to the test solution preparation method. The average recovery rate was calculated to be 97.57%, and the RSD was 4.77%. The results are shown in Table 9.

[0116] Table 9 Results of Puerarin Recovery Test

[0117] 1 392.5 314 694.2 96.08 2 390.8 314 697.6 97.71 3 393.2 314 699.8 97.64 4 391.6 392.5 778.5 98.57 5 392 392.5 768.3 95.87 6 392.8 392.5 772.4 96.71 7 391.2 471 857.2 98.94 8 390.5 471 849.8 97.52 9 392.3 471 858.7 99.02

[0118] 9. Content determination of three batches of samples

[0119] Three batches of samples were taken, and the puerarin content was determined according to the above method. The results are shown in Table 10.

[0120] Table 10 Results of Puerarin Content Determination in Three Batches of Samples

[0121] 20201001 4.12 82.4 20201002 3.89 77.8 20201003 4.01 80.2 mean 4.005 80.1 RSD (%) 3.54 3.54

[0122] Example 6: Stability Test

[0123] 1. Long-term stability test

[0124] Three batches of samples (batch numbers 20201001, 20201002, and 20201003) were collected and stored at room temperature (25℃±2℃) and relative humidity (60%±10%) for 12 months (October 2020 to October 2021). Samples were taken at 0, 3, 6, 9, and 12 months to investigate their properties, TLC identification, particle size, moisture content, solubility, fill weight variation, puerarin content, and microbial limits. The results are shown in Table 11.

[0125] Table 11 Results of Long-Term Stability Test (Batch No. 20201001)

[0126] 0 brown granules 9.2 3.6 Compliant 82.4 Compliant 3 brown granules 9.3 3.8 Compliant 81.6 Compliant 6 brown granules 9.5 4 Compliant 80.9 Compliant 9 brown granules 9.4 4.2 Compliant 79.5 Compliant 12 brown granules 9.6 4.3 Compliant 78.2 Compliant

[0127] The long-term stability test results of the three batches of samples showed a consistent trend, with no significant changes in any of the indicators and no significant decrease in puerarin content.

[0128] 2. Accelerated stability test

[0129] Three batches of samples were taken and placed in a constant temperature and humidity incubator (40℃±2℃, relative humidity 75%±5%) for 6 months. Samples were taken at 0, 1, 2, 3 and 6 months to examine various indicators. The results are shown in Table 12.

[0130] Table 12 Accelerated stability test results (batch number 20201001)

[0131] 0 brown granules 9.2 3.6 Compliant 82.4 Compliant 1 brown granules 9.4 3.9 Compliant 81.7 Compliant 2 brown granules 9.6 4.1 Compliant 80.5 Compliant 3 brown granules 9.5 4.3 Compliant 79.8 Compliant 6 brown granules 9.8 4.5 Compliant 78.6 Compliant

[0132] The accelerated stability test results of the three batches of samples showed a consistent trend, with no significant changes in any of the indicators.

[0133] Based on the combined results of long-term and accelerated stability tests, the shelf life of this product is tentatively set at 24 months.

[0134] Example 7: Anticoagulation and Hemorheology Experiments in Normal Animals

[0135] 1. Experimental Materials

[0136] 1.1 Laboratory Animals

[0137] ICR mice, male, weighing 18–22 g, SPF grade. SD rats, male, weighing 220–250 g, SPF grade.

[0138] 1.2 Experimental Drugs

[0139] Yinao Tongluo Granules (batch number 20201001) were prepared according to the method in Example 1 of this invention, and were prepared with distilled water to the required concentration before use.

[0140] 2. Measurement of bleeding time in mice

[0141] 2.1 Animal grouping and administration

[0142] Forty ICR mice were randomly divided into four groups of 10 mice each: a blank control group (distilled water), a low-dose group (0.5 g / kg), a medium-dose group (1.0 g / kg), and a high-dose group (2.0 g / kg) of Yinaotongluo granules. Each group was administered the medication by gavage once daily for seven consecutive days.

[0143] 2.2 Measurement Method

[0144] One hour after the last administration, the mice were fixed and the tip of their tails was cut off by about 3 mm. After the blood flowed out on its own, the mice were gently wiped with filter paper every 10 seconds until there was no blood left on the filter paper. The bleeding time was recorded (capillary method).

[0145] 2.3 Experimental Results

[0146] The results are shown in Table 13.

[0147] Table 13 Effect of Yinao Tongluo Granules on Bleeding Time in Mice (Mean±SD, n=10)

[0148] Blank control group — 68.4 ± 12.6 low-dose group 0.5 79.2 ± 14.3 medium dose group 1 93.7 ± 15.8* High-dose group 2 114.5 ± 18.2**

[0149] Note: Compared with the blank control group, *P<0.05, **P<0.01.

[0150] The results showed that the medium and high doses of Yinaotongluo granules significantly prolonged the bleeding time in mice in a dose-dependent manner.

[0151] 3. Mouse coagulation time determination

[0152] 3.1 Animal grouping and administration

[0153] Same as the bleeding time measurement item.

[0154] 3.2 Measurement Method

[0155] One hour after the last administration, blood was collected from the retrocanal venous plexus of the mouse eye using a capillary tube, and the timing was immediately recorded. Every 10 seconds, one end of the capillary tube was broken by about 0.5 cm to observe whether blood clots appeared, and the clotting time was recorded.

[0156] 3.3 Experimental Results

[0157] The results are shown in Table 14.

[0158] Table 14 Effect of Yinao Tongluo Granules on Coagulation Time in Mice (Mean±SD, n=10)

[0159] Blank control group — 92.6 ± 15.4 low-dose group 0.5 103.8 ± 16.7 medium dose group 1 121.3 ± 18.5* High-dose group 2 148.7 ± 22.6**

[0160] Note: Compared with the blank control group, *P<0.05, **P<0.01.

[0161] The results showed that the medium and high doses of Yinaotongluo granules significantly prolonged the coagulation time of mice in a dose-dependent manner.

[0162] 4. Rat coagulation assay

[0163] 4.1 Animal grouping and administration

[0164] Forty SD rats were randomly divided into four groups of 10 rats each: a blank control group (distilled water), a low-dose group (0.4 g / kg), a medium-dose group (0.8 g / kg), and a high-dose group (1.6 g / kg) of Yinaotongluo granules. Each group was administered the medication by gavage once daily for seven consecutive days.

[0165] 4.2 Determination Method

[0166] One hour after the last administration, anesthesia was achieved by intraperitoneal injection of sodium pentobarbital (40 mg / kg). Blood was drawn from the abdominal aorta and anticoagulated with 3.8% sodium citrate (1:9). The plasma was separated by centrifugation at 3000 rpm for 15 minutes. The activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB) levels were measured using an automated coagulation analyzer.

[0167] 4.3 Experimental Results

[0168] The results are shown in Table 15.

[0169] Table 15 Effects of Yinao Tongluo Granules on Coagulation Parameters in Rats (Mean±SD, n=10)

[0170] Blank control group — 18.6 ± 2.1 12.4 ± 1.3 22.5 ± 2.8 2.86 ± 0.32 low-dose group 0.4 20.3 ± 2.4 13.1 ± 1.4 24.2 ± 2.6 2.71 ± 0.28 medium dose group 0.8 23.8 ± 2.8* 14.6 ± 1.6* 27.8 ± 3.1* 2.43 ± 0.26* High-dose group 1.6 27.5 ± 3.2** 16.3 ± 1.8** 32.4 ± 3.6** 2.12 ± 0.24**

[0171] Note: Compared with the blank control group, *P<0.05, **P<0.01.

[0172] The results showed that the medium and high dose groups of Yinaotongluo granules could significantly prolong APTT, PT, and TT, and reduce FIB content, thus exhibiting a clear anticoagulant effect.

[0173] Example 8: Pharmacodynamic Experiment of Rats with Cerebral Ischemia-Reperfusion Injury

[0174] 1. Experimental Materials

[0175] 1.1 Laboratory Animals

[0176] SD rats, male, weighing 240–260 g, SPF grade.

[0177] 1.2 Experimental Drugs

[0178] Yinao Tongluo Granules (batch number 20201001), prepared according to the method of Example 1 of this invention. Ginkgo Leaf Drops (positive control drug).

[0179] 2. Animal grouping and administration

[0180] Seventy SD rats were randomly divided into seven groups of ten each: sham-operated group, blood stasis group, blood stasis model group, ginkgo leaf group (0.2 g / kg), low-dose Yinaotongluo granules group (0.4 g / kg), medium-dose group (0.8 g / kg), and high-dose group (1.6 g / kg).

[0181] After 7 days of acclimatization, each group was administered the drug once daily by gavage for 7 consecutive days. The sham surgery group, blood stasis group, and blood stasis model group were administered an equal volume of distilled water by gavage. The model was established 1 hour after the last administration.

[0182] 3. Model Building

[0183] An acute blood stasis model combined with a middle cerebral artery occlusion / reperfusion (MCAO / R) model was used.

[0184] 3.1 Acute Blood Stasis Model

[0185] On day 1 of modeling, rats were subcutaneously injected with adrenaline (0.8 mg / kg) twice (4 hours apart). After each injection, the rats were immersed in ice water at 0-2°C for 5 minutes. The same treatment was performed on day 2.

[0186] 3.2 MCAO / R Model

[0187] After the blood stasis model was established, the MCAO / R model was established using the suture occlusion method. Rats were anesthetized with sodium pentobarbital (40 mg / kg) via intraperitoneal injection. A midline incision was made in the neck, and the right common carotid artery, external carotid artery, and internal carotid artery were separated. A suture occluded the blood flow to the middle cerebral artery by inserting it into the internal carotid artery through the external carotid artery. After 2 hours of occlusion, the suture occluded the rats, and reperfusion was restored for 24 hours. In the sham-operated group and the blood stasis group, only the common carotid artery was separated, without inserting a suture occluder.

[0188] 4. Detection Indicators and Methods

[0189] 4.1 Measurement of brain water content

[0190] Twenty-four hours after reperfusion, the rats in each group were decapitated, and their brains were harvested. The wet weight was measured, and the brains were dried in an oven at 105°C until constant weight. The brain water content was calculated using the following formula: Brain water content (%) = (Wet weight - Dry weight) / Wet weight × 100%

[0191] 4.2 Blood rheology measurement

[0192] Blood was drawn from the abdominal aorta (anticoagulated with sodium heparin), and whole blood viscosity (low, medium, and high shear rates) and plasma viscosity were measured using a fully automated blood rheometer.

[0193] 4.3 Measurement of Erythrocyte-Related Parameters

[0194] EDTA-K2 anticoagulated blood was analyzed using a fully automated hematology analyzer, which measured hematocrit (HCT), aggregation index, rigidity index, deformability index, and electrophoretic index.

[0195] 4.4 Coagulation panel test

[0196] Sodium citrate was used as an anticoagulant. Plasma was collected by centrifugation, and APTT, PT, TT, and FIB were measured using a fully automated coagulation analyzer.

[0197] 5. Statistical processing

[0198] Data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0199] 6. Experimental Results

[0200] 6.1 Effects on brain water content

[0201] The results are shown in Table 16.

[0202] Table 16 Effect of Yinao Tongluo Granules on Brain Water Content in MCAO / R Rats (Mean±SD, n=10)

[0203] Sham surgery group — 77.2 ± 1.3 Blood stasis group — 77.5 ± 1.4 Blood stasis model group — 82.6 ± 2.1** Ginkgo leaf group 0.2 78.8 ± 1.6**## YN low 0.4 80.3 ± 1.8*# YN 0.8 79.1 ± 1.5**## YN High 1.6 78.2 ± 1.4**##

[0204] Note: Compared with the sham surgery group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0205] The results showed that, compared with the sham-operated group, the brain water content in the blood stasis model group was significantly increased (P<0.01), indicating that the cerebral edema model was successfully established. Compared with the model group, the brain water content in all dose groups of Yinaotongluo granules was significantly reduced in a dose-dependent manner, and the high-dose group had an effect comparable to that of the Ginkgo biloba group.

[0206] 6.2 Effects on blood rheology

[0207] The results are shown in Table 17.

[0208] Table 17 Effects of Yinao Tongluo Granules on whole blood viscosity and plasma viscosity in MCAO / R rats (Mean±SD, n=10)

[0209] Sham surgery group 7.82 ± 0.86 4.16 ± 0.45 3.41 ± 0.32 1.28 ± 0.12 Blood stasis group 8.12 ± 0.92 4.28 ± 0.48 3.52 ± 0.35 1.31 ± 0.13 Blood stasis model group 12.86 ± 1.34** 5.92 ± 0.62** 4.56 ± 0.48** 1.82 ± 0.18** Ginkgo leaf group 9.23 ± 1.02**## 4.58 ± 0.52**## 3.68 ± 0.38**## 1.42 ± 0.14**## YN low 11.24 ± 1.18*# 5.32 ± 0.56*# 4.18 ± 0.42*# 1.68 ± 0.16*# YN 10.16 ± 1.08**## 4.86 ± 0.50**## 3.86 ± 0.40**## 1.52 ± 0.14**## YN High 9.02 ± 0.96**## 4.52 ± 0.48**## 3.62 ± 0.36**## 1.38 ± 0.13**##

[0210] Note: Compared with the sham surgery group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0211] The results showed that, compared with the model group, the whole blood viscosity and plasma viscosity of Yinaotongluo granules were significantly reduced in each dose group, showing a dose-dependent effect.

[0212] 6.3 Effects on erythrocyte parameters

[0213] The results are shown in Table 18.

[0214] Table 18 Effects of Yinao Tongluo Granules on Erythrocyte Parameters in MCAO / R Rats (Mean±SD, n=10)

[0215] Sham surgery group 42.6 ± 3.2 2.58 ± 0.24 3.86 ± 0.32 0.68 ± 0.06 5.42 ± 0.48 Blood stasis group 43.8 ± 3.5 2.72 ± 0.26 4.02 ± 0.35 0.65 ± 0.06 5.28 ± 0.46 Blood stasis model group 52.4 ± 4.6** 3.86 ± 0.38** 5.28 ± 0.48** 0.42 ± 0.04** 3.86 ± 0.36** Ginkgo leaf group 45.2 ± 3.8**## 2.96 ± 0.28**## 4.22 ± 0.38**## 0.62 ± 0.06**## 5.02 ± 0.44**## YN low 48.6 ± 4.2*# 3.42 ± 0.32*# 4.82 ± 0.42*# 0.48 ± 0.05*# 4.28 ± 0.40*# YN 46.8 ± 4.0**## 3.08 ± 0.30**## 4.46 ± 0.40**## 0.56 ± 0.05**## 4.72 ± 0.42**## YN High 44.6 ± 3.6**## 2.86 ± 0.26**## 4.18 ± 0.36**## 0.64 ± 0.06**## 5.08 ± 0.46**##

[0216] Note: Compared with the sham surgery group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0217] The results showed that, compared with the model group, the HCT, aggregation index, and rigidity index of Yinaotongluo granules were significantly reduced in each dose group, while the deformation index and electrophoretic index were significantly increased, showing a dose-dependent effect.

[0218] 6.4 Effects on coagulation parameters

[0219] The results are shown in Table 19.

[0220] Table 19 Effects of Yinao Tongluo Granules on Coagulation Parameters in MCAO / R Rats (Mean±SD, n=10)

[0221] Sham surgery group 18.2 ± 2.0 12.2 ± 1.2 22.0 ± 2.6 2.82 ± 0.30 Blood stasis group 17.6 ± 1.8 11.8 ± 1.1 21.2 ± 2.4 2.96 ± 0.32 Model group 14.8 ± 1.6** 10.2 ± 1.0** 18.4 ± 2.2** 3.68 ± 0.38** Ginkgo leaf group 21.2 ± 2.4**## 14.6 ± 1.5**## 26.8 ± 2.8**## 2.46 ± 0.26**## YN low 16.8 ± 1.8*# 11.6 ± 1.2*# 20.8 ± 2.4*# 3.28 ± 0.34*# YN 19.6 ± 2.2**## 13.6 ± 1.4**## 24.2 ± 2.6**## 2.68 ± 0.28**## YN High 22.4 ± 2.6**## 15.2 ± 1.6**## 28.6 ± 3.0**## 2.38 ± 0.24**##

[0222] Note: Compared with the sham surgery group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0223] The results showed that, compared with the model group, the medium and high dose groups of Yinaotongluo granules significantly prolonged APTT, PT, and TT, and reduced FIB content.

[0224] Example 9: Determination of neurological function and cerebral infarction volume in rats with cerebral ischemia-reperfusion injury

[0225] Experimental animals: Male SD rats

[0226] The experimental drug was the Yinao Tongluo Granules of this invention.

[0227] Experimental Materials and Grouping

[0228] Seventy SD rats were randomly divided into seven groups of ten each: sham-operated group, model group, deferiphenone tablet group (positive control group), ginkgo leaf group (positive control group), low-dose Yinaotongluo granules group (YN low), medium-dose Yinaotongluo granules group (YN medium), and high-dose Yinaotongluo granules group (YN high).

[0229] Twenty-four hours after reperfusion, the degree of neurological impairment in each group of rats was assessed using the modified rat neurological deficit severity score (mNSS). The mNSS score includes multiple tests such as motor, sensory, reflex, and balance, with a total score of 0-18 points. A higher score indicates a more severe neurological deficit.

[0230] After neurological function scoring, rats in each group were decapitated, and their brains were harvested, removing the olfactory bulb and cerebellum. The brains were then frozen at -20°C for 20 minutes. After removal, the brains were coronally sectioned into 2 mm thick slices and incubated in 2% TTC (2,3,5-triphenyltetrazolium chloride) solution at 37°C in the dark for 30 minutes. Normal brain tissue stained red, while infarcted areas appeared white. The percentage of cerebral infarction area was analyzed using ImageJ software. Cerebral infarction percentage (%) = (infarct area / total brain area) × 100%.

[0231] Data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0232] Impact on neurological deficit scores

[0233] The results are shown in Table 20.

[0234] Table 20 Effect of Yinao Tongluo Granules on mNSS score in MCAO / R rats (Mean±SD, n=10)

[0235] Sham surgery group — 0.0 ± 0.0 Model group — 12.8 ± 1.6** Deferone tablets 20mg / kg 7.2 ± 1.0**## Ginkgo leaf group 0.2g / kg 7.6 ± 1.2**## YN low 0.4g / kg 10.2 ± 1.4*# YN 0.8g / kg 8.6 ± 1.2**## YN High 1.6g / kg 7.2 ± 1.0**##

[0236] Note: Compared with the sham surgery group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0237] The results showed that the mNSS score in the model group was significantly higher than that in the sham surgery group (P<0.01), indicating that the neurological deficit model was successfully established. Compared with the model group, the mNSS scores of all dose groups of Yinaotongluo granules were significantly lower in a dose-dependent manner. The high-dose group was comparable to the deferiphenone tablet group and superior to the Ginkgo biloba group.

[0238] Example 10: Pathological observation of brain tissue in rats with cerebral ischemia-reperfusion injury

[0239] 1. Experimental Materials and Grouping

[0240] Same as Example 9.

[0241] Detection indicators and methods

[0242] Twenty-four hours after reperfusion, rats in each group had their brains removed after cardiac perfusion with physiological saline and fixation with 4% paraformaldehyde. The brains were then fixed in 4% paraformaldehyde for 24 hours, routinely dehydrated, embedded in paraffin, and sectioned (4 μm thick). HE staining and Nissl staining were performed, and the pathological morphological changes of the brain tissue were observed under a light microscope.

[0243] HE staining results (see) Figure 1 HE staining results of rats 24 h after ischemia-reperfusion (×200, ×400)

[0244] Sham-operated group: The neurons in the rat brain tissue were intact, with normal cell morphology, neat and dense arrangement, uniform cytoplasmic staining, clear nucleoli, and no obvious inflammatory cell infiltration.

[0245] Model group: The brain tissue showed obvious ischemic damage, disordered neuronal arrangement, significant cell swelling, atrophy of some neurons, nuclear condensation and nuclear dissolution, numerous vacuolar degeneration, widening of the interstitial spaces, and obvious inflammatory cell infiltration.

[0246] In the deferoxone tablet group: brain tissue pathological damage was significantly improved, neurons were arranged more neatly, cell morphology was close to normal, there was no obvious nuclear pyknosis and vacuolar degeneration, and the inflammatory response was significantly reduced.

[0247] Ginkgo leaf group: Brain tissue pathological damage was significantly reduced, neurons were more neatly arranged, cell morphology tended to be normal, nuclear pyknosis and vacuolar degeneration were significantly reduced, and inflammatory cell infiltration was significantly reduced.

[0248] Low-dose brain-nourishing and meridian-clearing group (YN low): The pathological damage to brain tissue was reduced compared with the model group. Some neurons were still relatively well arranged, but some neurons were still swollen and had nuclear condensation. Inflammatory cell infiltration was reduced.

[0249] In the medium-dose group of Yinaotongluo (YN medium): the pathological damage to brain tissue was significantly reduced, neurons were arranged more neatly, cell morphology tended to be normal, nuclear pyknosis and vacuolar degeneration were significantly reduced, and inflammatory cell infiltration was significantly reduced.

[0250] High-dose brain-nourishing and meridian-clearing group (YN high): Brain tissue pathological damage was significantly improved, neurons were neatly and tightly arranged, cell morphology was close to normal, nucleoli were clear, there was no obvious nuclear pyknosis and vacuolar degeneration, and the inflammatory response was significantly reduced, approaching the level of the sham surgery group.

[0251] Nissl staining results (see) Figure 2 Nissl staining results of rats 24 h after ischemia-reperfusion (×200, ×400)

[0252] Sham surgery group: The neurons in the cerebral cortex are rich in Nissl bodies, which are dark blue granular or clump-like and evenly distributed in the cytoplasm, with clear cell outlines.

[0253] Model group: The number of neurons in the cerebral cortex was significantly reduced, the number of Nissl bodies in the remaining neurons was significantly reduced, the staining was light or absent, the cell structure was destroyed, and the cell bodies were shrunken.

[0254] In the deferoxone tablet group: the number of Nissl bodies increased significantly, appearing dark blue, evenly distributed, with normal cell morphology, approaching the level of the sham-operated group.

[0255] Ginkgo leaf group: The number of Nissl bodies increased significantly, stained deep blue, and the cell morphology tended to be normal with clear outlines.

[0256] The low-dose group of Yinaotongluo (low YN): The number of Nissl bodies increased compared with the model group, and the staining was darker, but the cell morphology was still irregular.

[0257] In the brain-boosting and meridian-clearing medium-dose group (YN medium): the number of Nissl bodies increased significantly, the staining was deep blue, the cell morphology tended to be normal, and the outlines were clear.

[0258] High-dose brain-boosting and blood-circulating group (YN high): The number of Nissl bodies increased significantly, appearing dark blue, evenly distributed, with normal cell morphology, close to the level of the sham-operated group.

[0259] The above results indicate that the Yinao Tongluo Granules of the present invention can effectively reduce the pathological damage to brain tissue after cerebral ischemia-reperfusion injury, maintain the normal morphological structure of neurons, and exhibit dose-dependent effects.

[0260] Example 11: Detection of Oxidative Stress and Inflammatory Markers in Rats with Cerebral Ischemia-Reperfusion Injury

[0261] 1. Experimental Materials and Grouping

[0262] Same as Example 8.

[0263] 2. Model Building

[0264] Same as Example 8.

[0265] 3. Detection Indicators and Methods

[0266] Twenty-four hours after reperfusion, rats in each group were decapitated and their brains were harvested. Brain tissue from the infarcted side was collected, and 9 times the volume of pre-cooled physiological saline was added. The tissue was homogenized in an ice bath to prepare a 10% brain tissue homogenate. The homogenate was centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected. The following indicators were measured according to the instructions of the corresponding biochemical reagent kits:

[0267] Superoxide dismutase (SOD) activity;

[0268] Malondialdehyde (MDA) content;

[0269] Ferrous ions (Fe) 2+ )content;

[0270] Glutathione (GSH) content;

[0271] Interleukin-1β (IL-1β) levels;

[0272] Tumor necrosis factor-α (TNF-α) levels;

[0273] Catalase (CAT) content;

[0274] NADPH / NADP + ratio.

[0275] 4. Experimental Results

[0276] The results are shown in Table 21.

[0277] Table 21 Effects of Yinao Tongluo Granules on Oxidative Stress and Inflammatory Markers in Brain Tissue of MCAO / R Rats (Mean±SD, n=10)

[0278] Sham surgery group 86.4±8.2 2.86±0.32 0.86±0.10 12.8±1.4 42.6±4.2 1.86±0.18 18.6±2.4 26.4±3.2 Model group 42.6±4.6** 7.24±0.78** 2.68±0.28** 5.2±0.6** 22.4±2.6** 0.86±0.10** 58.6±6.4** 72.8±8.2** Ginkgo leaf group 72.8±7.6**## 3.86±0.42**## 1.28±0.14**## 10.4±1.2**## 36.8±3.8**## 1.62±0.16**## 26.8±3.2**## 34.6±4.0**## YN low 52.4±5.6**# 5.86±0.62**# 2.02±0.22**# 6.8±0.8**# 26.8±3.0**# 1.08±0.12**# 46.4±5.2**# 58.2±6.4**# YN 64.8±6.8**## 4.42±0.48**## 1.52±0.16**## 8.6±1.0**## 32.4±3.4**## 1.42±0.14**## 34.2±4.0**## 42.6±5.0**## YN High 76.2±8.0**## 3.42±0.38**## 1.16±0.12**## 11.2±1.2**## 38.2±4.0**## 1.68±0.18**## 24.6±3.0**## 32.4±3.8**##

[0279] Note: Compared with the sham surgery group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0280] The results showed that, compared with the sham-operated group, the model group had lower levels of oxidation products MDA and Fe in its brain tissue. 2+ The levels were significantly increased, along with the activities of antioxidant enzymes SOD, GSH, and CAT, and the NADPH / NADP ratio. + The ratio decreased significantly, while the levels of pro-inflammatory factors IL-1β and TNF-α increased significantly (P<0.01). After intervention with the granules of this invention, all the above indicators were significantly reversed in a dose-dependent manner, with the high-dose group showing better improvement than the Ginkgo biloba group. The results indicate that this invention can effectively inhibit oxidative stress damage in brain tissue, reduce inflammatory response, and correct redox imbalance.

[0281] Example 12: Detection of ferroptosis pathway protein expression in rats with cerebral ischemia-reperfusion injury

[0282] 1. Experimental Materials and Grouping

[0283] Same as Example 9.

[0284] 2. Detection Indicators and Methods

[0285] Western blot analysis

[0286] Twenty-four hours after reperfusion, rats in each group were decapitated and their brains were harvested. Brain tissue from the infarcted side was collected, and total protein was extracted using RIPA lysis buffer. Protein concentration was determined by the BCA method. Equal volumes of protein samples were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder for 1 hour, and then incubated overnight at 4°C with primary antibodies (GPX4, SLC7A11, NRF2, HO-1, and β-actin, all diluted 1:1000). The following day, after washing, the membranes were incubated with the corresponding secondary antibodies (1:5000) at room temperature for 1 hour, followed by ECL chemiluminescence imaging. Using β-actin as an internal control, the grayscale values ​​of the bands were analyzed using ImageJ software, and the relative expression levels of each protein were calculated (see [link to image]). Figure 3 Western blot images of NRF2, HO-1, SLC7A11, and GPX4 in the brain tissue of rats in each group; A: sham-operated group, B: model group, C: deferiphenone tablet group, D: Ginkgo biloba leaf group, E: low YN, F: medium YN, G: high YN).

[0287] Experimental results

[0288] Western blot results

[0289] The results are shown in Table 22.

[0290] Table 22 Effects of Yinao Tongluo Granules on the expression of GPX4, SLC7A11, NRF2, and HO-1 proteins in the brain tissue of MCAO / R rats

[0291] Group GPX4 / β-actin SLC7A11 / β-actin NRF2 / β-actin HO-1 / β-actin Sham surgery group 1.36 ± 0.04 1.31 ± 0.03 0.34 ± 0.01 0.23 ± 0.01 Model group 0.38 ± 0.01** 0.39 ± 0.02** 0.56 ± 0.01** 0.48 ± 0.01** Deferone tablets 0.99 ± 0.02**## 0.93 ± 0.02**## 0.95 ± 0.04**## 0.92 ± 0.05**## Ginkgo leaf group 0.91 ± 0.02**## 0.89 ± 0.01**## 1.02 ± 0.08**## 0.98 ± 0.05**## YN low 0.63 ± 0.02**## 0.56 ± 0.03**## 0.79 ± 0.02**## 0.78 ± 0.03**## YN 0.91 ± 0.02**## 0.91 ± 0.01**## 0.92 ± 0.05**## 0.97 ± 0.02**## YN High 1.18 ± 0.03**## 1.27 ± 0.02**## 1.26 ± 0.03**## 1.29 ± 0.02**##

[0292] Note: Compared with the sham surgery group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0293] Western blot results analysis

[0294] NRF2 protein expression: Compared with the sham-operated group, the NRF2 protein expression level in the model group rats was significantly increased (P<0.01), suggesting that the body initiates an endogenous antioxidant response after cerebral ischemia-reperfusion injury. Compared with the model group, the NRF2 protein expression in the deferiphene tablet group and the Ginkgo biloba control group was significantly increased (P<0.01); the NRF2 protein expression in the low-dose Yinaotongluo group was also significantly higher than that in the model group (P<0.05), and the medium-dose group showed a further increase (P<0.01), approaching the levels of the deferiphene tablet and Ginkgo biloba groups, while the high-dose group showed the highest NRF2 protein expression.

[0295] HO-1 protein expression: Compared with the sham-operated group, HO-1 protein expression was significantly increased in the model group (P<0.01). Compared with the model group, HO-1 protein expression was significantly increased in all drug-treated groups (P<0.01), with the Yinaotongluo group showing a dose-dependent increasing trend, and the high-dose group showing the highest HO-1 expression.

[0296] SLC7A11 protein expression: Compared with the sham-operated group, SLC7A11 protein expression in the model group was significantly reduced (P<0.01). Among the drug administration groups, the positive expression of SLC7A11 in the deferiphenone tablet group, the Ginkgo biloba control group, and the low, medium, and high dose groups of Yinaotongluo were significantly higher than that in the model group (P<0.01), and the Yinaotongluo group showed a dose-dependent increasing trend. The SLC7A11 expression in the high-dose group (1.27 ± 0.02) was close to the level of the sham-operated group (1.31 ± 0.03).

[0297] GPX4 protein expression: Compared with the sham-operated group, GPX4 protein expression in the model group was significantly reduced (P<0.01). Among the drug administration groups, the positive expression of GPX4 in the deferiphenone tablet group, the Ginkgo biloba control group, and the low, medium, and high dose groups of Yinaotongluo were significantly higher than that in the model group (P<0.01), and the Yinaotongluo group showed a dose-dependent increasing trend. The GPX4 expression in the high-dose group (1.18 ± 0.03) was close to the level of the sham-operated group (1.36 ± 0.04).

[0298] In summary, this invention discloses a brain-boosting and meridian-clearing traditional Chinese medicine composition and its preparation method. Addressing the technical shortcomings of traditional decoctions composed of renowned traditional Chinese medicine formulas (such as Leonurus japonicus, processed Polygonum multiflorum, Pueraria lobata, Ligusticum chuanxiong, Eucommia ulmoides, Carthamus tinctorius, and Alisma plantago-aquatica)—including inconvenience of administration, difficulty in industrial granulation due to the high hygroscopicity of extracts, insufficient quality control, and inadequate elucidation of the mechanism of action—this invention, through systematic screening of excipients and optimization of the entire process parameters, develops an oral solid dosage form that meets the granule standards of the Chinese Pharmacopoeia. A complete quality control system is established, including thin-layer chromatography qualitative identification, high-performance liquid chromatography content determination, and multi-dimensional routine inspections. Furthermore, a comprehensive animal experiment system verifies that this granule can improve hemodynamics, resist oxidative stress, and activate the NRF2 pathway to upregulate GPX4 and SLC7A11. The triple-linked neuroprotective mechanism of protein expression targeting and inhibiting ferroptosis in nerve cells has not only achieved the standardization and industrialization of this empirical formula, improving patient medication adherence and batch-to-batch quality stability, but also provided a novel Chinese medicine preparation with clear efficacy and controllable quality for the clinical prevention and treatment of ischemic stroke.

[0299] It should be understood that numerous specific implementation decisions can be made during the development of any practical implementation, such as in any engineering or design project. Such development efforts may be complex and time-consuming, but for those skilled in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.

[0300] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A brain-boosting and meridian-clearing traditional Chinese medicine granule, characterized in that, It includes active raw materials of traditional Chinese medicine and pharmaceutical excipients; the active raw materials are composed of the following components by weight: 20-25 parts of Leonurus japonicus, 12-17 parts of processed Polygonum multiflorum, 12-17 parts of Pueraria lobata, 12-17 parts of Ligusticum chuanxiong, 12-17 parts of Eucommia ulmoides, 9-13 parts of Carthamus tinctorius, and 5-9 parts of Alisma plantago-aquatica.

2. The traditional Chinese medicine granules according to claim 1, characterized in that: The active pharmaceutical ingredient, by weight, consists of the following components: 22.2 parts of Leonurus japonicus, 14.8 parts of processed Polygonum multiflorum, 14.8 parts of Pueraria lobata, 14.8 parts of Ligusticum chuanxiong, 14.8 parts of Eucommia ulmoides, 11.1 parts of Carthamus tinctorius, and 7.4 parts of Alisma plantago-aquatica.

3. The traditional Chinese medicine granules according to claim 1, characterized in that: The pharmaceutical excipient is soluble starch; the mass ratio of the dry extract powder of the active pharmaceutical ingredient to the soluble starch is 1:

1.

4. The method for preparing traditional Chinese medicine granules according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Raw material pretreatment: Weigh each raw material according to the prescription amount, clean and select the processed Polygonum multiflorum, Eucommia ulmoides, and Alisma plantago-aquatica, crush them into 10-mesh coarse powder, and cut the remaining medicinal materials into slices; soak them in water until they are saturated with water. S2. Water decoction extraction: The soaked medicinal materials are heated and refluxed twice for extraction, and the extracts are combined and filtered to remove residue; S3. Vacuum Concentration: The extract is concentrated under reduced pressure to a clear paste with a relative density of 1.25 to 1.35 as measured by thermal analysis at 80°C; S4. Drying and powdering: Dry the paste under reduced pressure to constant weight, pulverize and sieve to obtain dry paste powder; S5. Wet granulation: Mix the dry powder with soluble starch evenly, and prepare wet granules using ethanol as a wetting agent; S6. Drying and granulation: After drying the wet granules, granulate, mix, and package them to obtain the final product.

5. The method according to claim 4, characterized in that: Add 6 to 10 times the amount of water to S1 and soak for 50 minutes.

6. The method according to claim 4, characterized in that: In S2, the first extraction was carried out with 8 times the amount of water for 1.5 hours, and the second extraction was carried out with 6 times the amount of water for 1.0 hour. The extract was filtered through a 200-mesh sieve to remove residue.

7. The method according to claim 4, characterized in that: In S3, the vacuum concentration temperature is 80℃ and the vacuum degree is -0.08MPa; in S4, the vacuum drying temperature is 70℃ and the vacuum degree is -0.08MPa, and after drying, the product is pulverized through a 100-mesh sieve.

8. The method according to claim 4, characterized in that: In S5, the dry powder and soluble starch are added in equal amounts at a mass ratio of 1:1 and mixed evenly. The wetting agent is 75% ethanol by volume, and the amount used is 12% of the total mass of the powder. Granulation is carried out using a 16-mesh sieve. In S6, the drying temperature of the wet granules is 60℃ and the drying time is 3 hours. After drying, the granules are sieved through a 10-mesh sieve and an 80-mesh sieve for granulation. The mixture is mixed for 15-30 minutes and then sealed and packaged in 20g bags.

9. The use of the brain-nourishing and meridian-clearing traditional Chinese medicine granules according to any one of claims 1 to 3 in the preparation of drugs for the prevention and treatment of ischemic stroke.

10. The application according to claim 9, characterized in that, The drug is used to improve hemodynamics in ischemic brain regions, reduce oxidative stress damage in brain tissue, or inhibit ferroptosis in brain nerve cells. The drug activates the NRF2 signaling pathway, upregulates the expression of GPX4 and SLC7A11 proteins, and blocks the ferroptosis pathway induced by cerebral ischemia-reperfusion injury.

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