Method for improving neurological function recovery after cerebral apoplexy by using preparation

Abstract

The invention discloses a method for improving neurological function recovery after cerebral apoplexy through a preparation, and relates to the technical field of biomedicine.The method comprises the steps of traditional Chinese medicine preparation preparation, dosage matching and recovery situation evaluation.The method for improving neurological function recovery after cerebral apoplexy through the preparation comprises the following specific operation steps that firstly, a traditional Chinese medicine composition for treatment is prepared; comprising an extract A and an extract B, and the extract A and the extract B are mixed to prepare a nano suspension. The preparation method has the advantages that fat-soluble and water-soluble effective components in a prescription are enriched in a targeted manner through a subsection extraction strategy of combining supercritical CO2 extraction and enzymolysis-ultrasonic-assisted water extraction, the limitation of a traditional single extraction method is overcome, and the preparation process is simple and convenient. The nano preparation with uniform particle size and high stability is successfully prepared by using a high-pressure homogeneous nanocrystallization technology, the bioavailability of the medicine is remarkably improved, and the potential of the medicine for crossing the blood brain barrier is enhanced.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for improving the recovery of neurological function after stroke through the formulation of a preparation. Background Technology

[0002] Ischemic stroke is a serious disease caused by the interruption of blood supply to the brain, leading to brain tissue hypoxia, necrosis and neurological deficits. Currently, clinical treatment mainly involves intravenous thrombolysis and endovascular thrombectomy, but these methods have strict time window limitations and bleeding risks, and the treatment options for the neurological recovery period are very limited.

[0003] The applicant discovered through a search that a Chinese patent, "Application of Xiangshao Granules in the Preparation of Drugs to Improve Neurological Deficiency After Stroke," with publication number "CN115475218A," mainly provides a new use for Xiangshao Granules. RT-PCR (quantitative real-time PCR) and ELISA (enzyme-linked immunosorbent assay) confirmed that it can reduce the expression levels of TNFα, IL6, and IL1β in tissues at the gene and protein levels, respectively. Western blot analysis showed that Xiangshao Granules inhibit neuroinflammation by acting on the TLR4-NFκb pathway and inhibit apoptosis of prefrontal and hippocampal neurons. Nissl staining confirmed that Xiangshao Granules can increase the number of hippocampal neurons, ultimately improving the behavior of stroke model mice. This indicates that Xiangshao Granules have the potential application in preparing drugs to improve neurological function after stroke. However, its mechanism of action is relatively simple. Therefore, we propose a method for improving neurological function recovery after stroke through formulation. Summary of the Invention

[0004] The purpose of this invention is to provide a method for improving the recovery of neurological function after stroke.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for improving the recovery of neurological function after stroke, comprising the preparation of a traditional Chinese medicine preparation, dosage ratio, and assessment of recovery status. The specific operation steps of the method for improving the recovery of neurological function after stroke are as follows:

[0006] Step 1: Prepare a traditional Chinese medicine composition for treatment, including extract A and extract B. Mix extract A and extract B to prepare a nano suspension.

[0007] Step 2: Administer the traditional Chinese medicine composition obtained in Step 1 orally to patients in the recovery period of stroke at a therapeutically effective dose;

[0008] Step 3: Use standardized neurological function rating scales to assess the patient's neurological function recovery.

[0009] As a further aspect of the present invention: in step one, the weight ratio of the raw materials is: Astragalus membranaceus 30 parts, Salvia miltiorrhiza 20 parts, Ligusticum chuanxiong 15 parts, Panax notoginseng 10 parts, and Hirudo medicinalis 5 parts. The preparation of the traditional Chinese medicine composition includes:

[0010] Supercritical CO2 extraction was performed on Astragalus membranaceus and Ligusticum chuanxiong in the raw materials to obtain extract A;

[0011] The raw materials, including Salvia miltiorrhiza, Panax notoginseng, and leeches, were subjected to biphasic hydrolysis and enzymatic hydrolysis, followed by ultrasonic-assisted water extraction, to obtain extract B.

[0012] Extract A and paste B are mixed and processed by high-pressure homogenization to prepare a nano suspension.

[0013] As a further aspect of the present invention: the pressure of the supercritical CO2 extraction is 25MPa-35MPa, and the temperature is 45℃-55℃.

[0014] As a further aspect of the present invention: the enzymatic hydrolysis is carried out using a complex enzyme of cellulase and pectinase, and the ultrasonic power of the ultrasonic-assisted water extraction is 600W, and the temperature is 70℃-80℃.

[0015] As a further aspect of the present invention: the pressure of the high-pressure homogenization treatment is 80MPa-100MPa, and the average particle size of the resulting nano-suspension is less than 200nm.

[0016] As a further aspect of the present invention: the extract A and the extract B are mixed in a water bath at 60℃-65℃, and pre-emulsified using a high-speed shear disperser at a speed of 10000r / min-12000r / min to form a primary emulsion, and then homogenized under high pressure.

[0017] As a further aspect of the present invention: after high-pressure homogenization, the nano suspension is subjected to vacuum freeze-drying to obtain nano-preparations.

[0018] As a further aspect of the present invention: during the high-pressure homogenization process, the material temperature is controlled below 60°C through a circulating cooling system.

[0019] As a further aspect of the present invention: before the vacuum freeze-drying, a freeze-drying protectant is added to the nano suspension, wherein the freeze-drying protectant is 5% w / v mannitol.

[0020] Compared with the prior art, the beneficial effects of the present invention by adopting the above technical solution are as follows:

[0021] 1. This invention utilizes a segmented extraction strategy combining supercritical CO2 extraction and enzymatic hydrolysis-ultrasound-assisted water extraction to selectively enrich both lipid-soluble and water-soluble active ingredients in the formulation, overcoming the limitations of traditional single extraction methods. Furthermore, by employing high-pressure homogenization nanotechnology, nano-formulations with uniform particle size and high stability are successfully prepared, significantly improving the bioavailability of the drug and enhancing its potential to cross the blood-brain barrier.

[0022] 2. This invention, through standardized modern extraction and nano-formulation technology, yields a traditional Chinese medicine composition with relatively well-defined components and controllable quality, which can clearly reveal the synergistic effects of this formulation on multiple pathways such as protecting the blood-brain barrier, inhibiting inflammatory responses, and promoting neurotrophic effects.

[0023] 3. This invention clearly provides the dosage scheme for clinical application of traditional Chinese medicine nano-preparations and the objective efficacy evaluation method based on internationally recognized scales, effectively solving the clinical dilemma of lacking specific drugs for the treatment of the recovery period, and has important clinical application value. Attached Figure Description

[0024] Figure 1 This is a flowchart of the method for restoring nerve function in an embodiment of the present invention. Detailed Implementation

[0025] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0026] Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0027] Please see the appendix Figure 1 This invention discloses a method for improving neurological function recovery after stroke, comprising the preparation of a traditional Chinese medicine preparation, dosage ratio, and assessment of recovery status. The specific operational steps of the method for improving neurological function recovery after stroke are as follows:

[0028] Step 1: Prepare a traditional Chinese medicine composition for treatment, including extract A and extract B. Mix extract A and extract B to prepare a nano suspension.

[0029] Step 2: Administer the traditional Chinese medicine composition obtained in Step 1 orally to patients in the recovery period of stroke at a therapeutically effective dose;

[0030] Step 3: Use standardized neurological function rating scales to assess the patient's neurological function recovery.

[0031] In one embodiment of the present invention: extract A is a fat-soluble oil and extract B is a water-soluble thick paste. They must be mixed in a water bath at 60°C-65°C and pre-emulsified using a high-speed shear disperser at a speed of 10000r / min-12000r / min for 5min-10min to form a preliminary emulsion. This is a prerequisite for successful high-pressure homogenization.

[0032] In one embodiment of the present invention: the high-pressure homogenization process generates a large amount of heat, which may lead to the degradation of heat-sensitive components. A circulating cooling system is configured in the high-pressure homogenization process to keep the material temperature below 60°C at all times.

[0033] In one embodiment of the present invention: after high-pressure homogenization and before freeze-drying, a freeze-drying protectant must be added to the nano suspension. The freeze-drying protectant is 5% (w / v) mannitol or sucrose. The protectant can form a stable glassy framework around the nanoparticles during freezing and drying, preventing particle aggregation and particle size increase, and ensuring that the original nano properties can be restored after reconstitution.

[0034] In one embodiment of the present invention: In step two, when the preparation is used in combination with antiplatelet drugs such as aspirin and clopidogrel, bleeding signs should be closely monitored. When used in combination with antihypertensive drugs, it is recommended to strengthen blood pressure monitoring in the early stage of treatment to prevent blood pressure from being too low. It is recommended that the time interval between taking this product and the above-mentioned Western medicines be 1-2 hours to reduce potential interactions.

[0035] Example

[0036] Drug preparation:

[0037] Take 3.0 kg of Astragalus membranaceus, 2.0 kg of Salvia miltiorrhiza, 1.5 kg of Ligusticum chuanxiong, 1.0 kg of Panax notoginseng, and 0.5 kg of Hirudo medicinalis;

[0038] After cleaning and drying, all medicinal materials were pulverized and passed through a 40-mesh sieve for later use. Astragalus and Ligusticum powders were thoroughly mixed and put into a 100L supercritical CO2 extraction vessel. The extraction pressure was set at 30MPa, the extraction temperature at 50℃, the pressure of analytical vessel I was 10MPa, the temperature at 60℃, the pressure of analytical vessel II was 5.5MPa, the temperature at 35℃, the CO2 flow rate was 300L / h, and the dynamic extraction was carried out for 2.5 hours.

[0039] Brownish-red oily extract A, totaling 1580g, was collected from analytical vessel II. HPLC analysis showed that the content of astragaloside A was 1.68% and the content of tetramethylpyrazine was 0.85%.

[0040] Preparation of extract B:

[0041] Add the powders of Salvia miltiorrhiza, Panax notoginseng, and Hirudo medicinalis to a 1000L multi-functional extraction tank, add 500L of purified water, stir and mix well, adjust the pH to 5.0 with citric acid solution, add 4.5kg of compound enzyme (cellulase: pectinase = 1:1), and gently stir and enzymatically hydrolyze for 1 hour at 48℃.

[0042] After enzymatic hydrolysis, turn on the ultrasonic system, set the power to 6000W, heat to 75℃, keep warm for 30 minutes, filter, add 400L of purified water to the residue, and perform a second ultrasonic extraction for 30 minutes under the same conditions.

[0043] The two filtrates were combined and concentrated under reduced pressure using a triple-effect concentrator at 60℃ and -0.09MPa.

[0044] A brownish-black extract B was obtained, totaling 15.2 kg. HPLC analysis showed that the content of salvianolic acid B was 5.5%, and the content of notoginsenoside R1 was 2.2%.

[0045] Take 1.58 kg of extract A and 15.2 kg of extract B, mix them in a 60℃ water bath, add 0.5 kg of soybean lecithin, and pre-emulsify them for 10 min at 10000 rpm using a high-speed shear disperser to obtain the colostrum;

[0046] The colostrum was passed through an industrial high-pressure homogenizer and homogenized 12 times under a pressure of 90MPa. During the homogenization process, the material temperature was controlled to remain below 60℃ through a cooling system.

[0047] Add 5% (w / v) mannitol to the above nano suspension, stir to dissolve, and then dispense into freeze-drying trays;

[0048] Freeze-drying parameters: Pre-freeze: -45℃, 3 hours; Main dry: -25℃ to 0℃, 24 hours; Vacuum degree <10Pa; Desorption dry: 0℃ to 25℃, 6 hours.

[0049] Results and data: Loose nanoparticles were obtained, with a reconstitution time of <30 seconds and a moisture content of 2.1%.

[0050] Experimental Design:

[0051] Animals: Healthy male SD rats, weighing 250±20g;

[0052] Grouping:

[0053] Sham surgery group (n=10);

[0054] Model control group (n=10);

[0055] The formulation group of this invention (n=10);

[0056] Positive control group (n=10);

[0057] Establishment of a stroke model:

[0058] A middle cerebral artery occlusion model was established using the suture occlusion method;

[0059] Ischemia time: 90 min;

[0060] Reperfusion time: 28 days;

[0061] Dosage regimen:

[0062] The formulation of this invention consists of nanoparticles, 100 mg / kg daily, administered by gavage.

[0063] Positive control group: Nimodipine, 10 mg / kg daily;

[0064] Model control group: an equal volume of physiological saline;

[0065] Dosage time: Begin 24 hours after reperfusion, for 28 consecutive days;

[0066] Neurological function recovery assessment:

[0067] Neurological deficit score (mNSS):

[0068] Time point Sham surgery group Model control group The formulation of the present invention Positive control group Day 1 0.2±0.1 12.5±1.2 12.3±1.1 12.4±1.3 Day 7 0.2±0.1 10.8±1.5 7.2±1.3*# 8.5±1.4* Day 14 0.2±0.1 9.2±1.6 4.5±1.2*# 6.3±1.5* Day 28 0.2±0.1 7.8±1.4 2.1±0.8*# 4.2±1.1*

[0069] Compared with the model group, P<0.05; # Compared with the positive control group, P<0.05;

[0070] Motor coordination ability test (rotating bar test):

[0071] Group Day 7 (s) Day 14 (s) Day 28 (s) Sham surgery group 185.2±15.3 186.5±14.8 187.2±15.1 Model control group 45.3±12.6 58.7±13.2 72.5±14.3 The formulation of the present invention 78.5±13.4*# 112.6±14.1*# 156.3±14.7*# Positive control group 65.2±12.8* 89.4±13.7* 125.8±14.2*

[0072] Cognitive function assessment (water maze test):

[0073] Group Escape from the 28th day of the incubation period (s) Platform crossing times Sham surgery group 12.5±3.2 6.8±1.5 Model control group 38.6±5.8 1.5±0.8 The formulation of the present invention 18.3±4.1*# 5.2±1.2*# Positive control group 25.7±4.6* 3.6±1.1*

[0074] Histological analysis:

[0075] Infarct volume:

[0076] TTC staining showed:

[0077] Model control group: 28.5±3.2%;

[0078] The formulation of this invention: 12.3±2.1%*#;

[0079] Positive control group: 18.6±2.7%*;

[0080] Neuron survival count:

[0081] Number of NeuN-positive cells in the peri-infarct area:

[0082] Sham surgery group: 125.6 ± 12.3 per field of view;

[0083] Model control group: 45.3±8.7 per field of view;

[0084] The formulation of this invention: 98.5 ± 10.2 cells / field of view*#;

[0085] Positive control group: 76.8±9.4 per field of view*;

[0086] Markers of synaptic plasticity:

[0087] Synaptophysin expression levels (Westernblot):

[0088] Model control group: 0.32±0.05;

[0089] The formulation group of this invention: 0.78±0.08*#;

[0090] Positive control group: 0.56±0.07*;

[0091] Blood-brain barrier permeability:

[0092] Evansblue exudate:

[0093] Model control group: 18.5±2.3μg / g;

[0094] The formulation of this invention: 8.2 ± 1.5 μg / g*#;

[0095] This invention demonstrates that the formulation can effectively protect the integrity of the blood-brain barrier;

[0096] Inflammatory factor levels:

[0097] TNF-α level (ELISA):

[0098] Model control group: 125.6±15.3 pg / mL;

[0099] The formulation of this invention: 65.8±12.4 pg / mL*#;

[0100] This indicates that the formulation of the present invention has significant anti-inflammatory effects;

[0101] Neurotrophic factors:

[0102] BDNF expression level:

[0103] Model control group: 0.45±0.06;

[0104] The formulation group of this invention: 0.92±0.09*#;

[0105] This indicates that the formulation of the present invention can promote the expression of neurotrophic factors;

[0106] Safety assessment:

[0107] General situation:

[0108] The rats in each group showed normal weight gain, no abnormal behavior, and good coat luster.

[0109] Blood biochemical indicators:

[0110] Liver and kidney function indicators were all within the normal range, and there were no abnormal changes in blood routine tests, indicating that the preparation of this invention has good safety at the experimental dose.

[0111] Establishment of a stroke model:

[0112] A mouse model of middle cerebral artery occlusion was established using the standard suture occlusion method approved by the animal ethics committee. The surgery was performed under isoflurane inhalation anesthesia. During the operation, the animal's body temperature was maintained at 37±0.5℃ using a heating pad. The operation time was strictly controlled within 30 minutes. Immediately after the operation, ibuprofen was administered for analgesia and the animal was placed in an incubator to recover.

[0113] Neurological function assessment methods:

[0114] All behavioral tests were conducted in a soundproof, well-lit laboratory environment by trained researchers who were unfamiliar with the group assignments, in order to eliminate subjective bias.

[0115] Modified Neurological Deficit Assessment (MDI)

[0116] The 18-point mNSS scoring system was used to assess motor, sensory, balance, and reflex functions. The assessment process was gentle and quick, with each assessment lasting no more than 10 minutes.

[0117] Rotating bar experiment:

[0118] Motor coordination was tested using a rotundimeter. Three days of acclimatization training were conducted before the experiment. During the formal test, the rotundimeter speed was gradually increased from 4 rpm to 40 rpm. The time the animal stayed on the rotundimeter was recorded. Soft padding was placed under the test platform to prevent the animal from falling and getting injured.

[0119] Morris Water Maze

[0120] The water temperature was maintained at 23±1℃, and fixed spatial references were placed around the pool. The experiment included a 5-day orientation navigation test and a 1-day space exploration test. After each test, the animals were immediately dried with a towel and put back into the insulated cage to prevent hypothermia.

[0121] Tissue sample collection:

[0122] After the experiment, the animals were subjected to cardiac perfusion under deep anesthesia to ensure that they did not suffer during the sampling process. The brain tissue samples were used for TTC staining to determine infarct volume and for immunohistochemical analysis.

[0123] Data Analysis:

[0124] All data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS 25.0 software. A p-value < 0.05 was considered statistically significant.

[0125] The embodiments fully demonstrate the specific implementation process and effects of the method of the present invention in improving the recovery of neurological function after stroke. The formulation process is stable and reliable, and the quality of the obtained nano-formulation meets the design requirements. Animal experiments have confirmed that the formulation can improve neurological deficit scores, enhance motor coordination, promote cognitive function recovery, reduce cerebral infarction volume, and increase neuronal survival. The mechanism of action involves multi-target regulation, including: protecting the blood-brain barrier, inhibiting inflammatory response, and promoting neurotrophic effects. This method provides an effective and safe treatment strategy for the recovery of neurological function after stroke and has important clinical application value.

[0126] The animal experiments described in this invention strictly adhere to ethical guidelines for the use of laboratory animals and have been reviewed and approved by the relevant animal ethics committees. Humanitarian principles are consistently upheld throughout the experiments to minimize animal suffering. This description is for the purpose of demonstrating the technical effectiveness of the invention only and does not constitute encouragement or promotion of animal experimentation.

[0127] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Any variations and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, any modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention, without departing from the scope of the invention, fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for improving neurological function recovery after stroke, comprising the preparation of a traditional Chinese medicine preparation, dosage ratio, and assessment of recovery status, characterized in that: The specific steps of the method for improving the recovery of neurological function after stroke using the aforementioned preparation are as follows: Step 1: Prepare a traditional Chinese medicine composition for treatment, including extract A and extract B. Mix extract A and extract B to prepare a nano suspension. Step 2: Administer the traditional Chinese medicine composition obtained in Step 1 orally to patients in the recovery period of stroke at a therapeutically effective dose; Step 3: Use standardized neurological function rating scales to assess the patient's neurological function recovery.

2. The method for improving neurological function recovery after stroke according to claim 1, characterized in that: In step one, the weight ratio of the raw materials is: Astragalus membranaceus 30 parts, Salvia miltiorrhiza 20 parts, Ligusticum chuanxiong 15 parts, Panax notoginseng 10 parts, and Hirudo medicinalis 5 parts. The preparation of the traditional Chinese medicine composition includes: Supercritical CO2 extraction was performed on Astragalus membranaceus and Ligusticum chuanxiong in the raw materials to obtain extract A; The raw materials, including Salvia miltiorrhiza, Panax notoginseng, and leeches, were subjected to biphasic hydrolysis and enzymatic hydrolysis, followed by ultrasonic-assisted water extraction, to obtain extract B. Extract A and paste B are mixed and processed by high-pressure homogenization to prepare a nano suspension.

3. The method for improving neurological function recovery after stroke according to claim 2, characterized in that: The supercritical CO2 extraction pressure is 25MPa-35MPa, and the temperature is 45℃-55℃.

4. A method for improving neurological function recovery after stroke according to claim 3, characterized in that: The enzymatic hydrolysis is carried out using a complex enzyme of cellulase and pectinase, and the ultrasonic-assisted water extraction has an ultrasonic power of 600W and a temperature of 70℃-80℃.

5. A method for improving neurological function recovery after stroke according to a formulation according to claim 4, characterized in that: The high-pressure homogenization process is carried out at a pressure of 80MPa-100MPa, and the resulting nano-suspension has an average particle size of less than 200nm.

6. A method for improving neurological function recovery after stroke according to a formulation according to claim 5, characterized in that: The extract A and the extract B are mixed in a water bath at 60℃-65℃ and pre-emulsified using a high-speed shear disperser at a speed of 10000r / min-12000r / min to form a primary emulsion before high-pressure homogenization.

7. A method for improving neurological function recovery after stroke according to a formulation according to claim 6, characterized in that: After high-pressure homogenization, the nano-suspension is freeze-dried under vacuum to obtain nano-formulations.

8. A method for improving neurological function recovery after stroke according to a formulation according to claim 7, characterized in that: During the high-pressure homogenization process, the material temperature is controlled below 60°C through a circulating cooling system.

9. A method for improving neurological function recovery after stroke according to a formulation according to claim 8, characterized in that: Before the vacuum freeze-drying, a freeze-drying protectant, namely 5% w / v mannitol, is added to the nano suspension.

Images