Traditional Chinese medicine composition for preventing and treating cerebral arterial thrombosis and screening method thereof

Abstract

The invention discloses a traditional Chinese medicine composition for preventing and treating cerebral arterial thrombosis and a screening method thereof, and the effective components of the traditional Chinese medicine composition comprise the following substances in parts by weight: 30-50 parts of 3-caffeoylquinic acid, 20-40 parts of 4, 5-dicaffeoylquinic acid and 30-50 parts of scutellarin. The traditional Chinese medicine composition disclosed by the invention has an obvious improvement effect on cerebral arterial thrombosis, and can also protect damaged neurons, improve the activity of nerve cells and effectively relieve cerebral arterial thrombosis injury. Meanwhile, according to the screening method of the composition, a regression model with brain protection related indexes as dependent variables and the proportion of all the components as independent variables is established through mixed material design, the predictive ability of the model is evaluated, and after the predictive ability of the model reaches the standard, synchronous optimization of the multiple brain protection indexes is carried out; and solving the optimal ratio of each component and carrying out experimental verification. The traditional Chinese medicine composition screened by the method is stable in activity and exact in effect.

Description

Technical Field

[0001] The present application relates to a Chinese medicine composition for preventing and treating ischemic stroke and a screening method thereof, belonging to the technical field of Chinese medicine. Background Art

[0002] Stroke is a major disease that threatens human health. It is the second leading cause of death after ischemic heart disease worldwide. Stroke is mainly divided into two categories: ischemic stroke caused by cerebral vascular obstruction and hemorrhagic stroke caused by cerebral vascular rupture. Among them, the number of patients with ischemic stroke accounts for 75%-85% of the total number of patients. However, due to the narrow treatment time window and complex pathogenesis, the prevention, health care and treatment effects of internal and external surgical methods and single-target Chinese medicine are not ideal. Therefore, it is of great significance to find a Chinese medicine composition with multi-component, multi-target therapeutic effects and overall conditioning effects in the prevention, health care and treatment of ischemic stroke.

[0003] Erigeron breviscapus (Vant.) Hand.-Mazz. is a herbaceous plant of the Asteraceae family mainly produced in Yunnan Province, China. It is named because its flowers resemble lanterns and its roots resemble asarum. It has the effects of promoting blood circulation, dredging collaterals, relieving pain, and dispelling wind and cold. It is often used to treat hemiplegia caused by stroke. Erigeron breviscapus is a multi-component natural Chinese medicine with complex chemical composition, mainly including caffeoyl components, flavonoids and their glycosides, volatile oil components and other compounds (such as coumarins, pentacyclic triterpenes, aromatic acids, phytosterols, xanthones, etc.). Current studies believe that flavonoid components and caffeoyl components are the main active ingredients of Erigeron breviscapus.

[0004] In recent years, a large number of studies have shown that flavonoid component scutellarin and caffeoyl components 3-caffeoylquinic acid, 4,5-dicaffeoylquinic acid, etc. can play a neuroprotective role by regulating mitochondrial homeostasis, maintaining intracellular and extracellular ion balance, protecting blood-brain barrier structure, anti-inflammatory, anti-oxidation, anti-apoptosis, etc. However, if the composition of scutellaria brevis extract is directly used, there are problems such as unclear effective substances and unknown mechanisms of action, which makes it difficult to obtain international recognition, and the secondary development of scutellaria brevis is limited; if only the role of a single component in scutellaria brevis is considered, it is contrary to the multi-biological process pathogenesis of cerebral ischemia / reperfusion injury, and the prevention, health care and treatment effects are bound to be poor; if the effective ingredients obtained from existing studies are made into a compound for research, it is difficult to scientifically determine the proportion of each component in the mixture, and it is impossible to obtain a Chinese medicine effective ingredient composition for the prevention, health care and treatment of ischemic stroke. Summary of the invention

[0005] According to one aspect of the present application, a Chinese medicine composition for preventing and treating ischemic stroke is provided, wherein the Chinese medicine composition is used to protect damaged neurons, increase nerve cell activity, and effectively reduce ischemic stroke damage.

[0006] A traditional Chinese medicine composition for preventing and treating ischemic stroke, characterized in that the effective ingredients of the traditional Chinese medicine composition include the following substances in parts by weight: 30-50 parts of 3-caffeoylquinic acid, 20-40 parts of 4,5-dicaffeoylquinic acid and 30-50 parts of scutellarin.

[0007] Furthermore, the effective ingredients of the traditional Chinese medicine composition include the following substances in parts by weight: 33 parts of 3-caffeoylquinic acid, 28 parts of 4,5-dicaffeoylquinic acid, and 39 parts of scutellarin;

[0008] Preferably, the active ingredient is derived from Erigeron breviscapus.

[0009] Furthermore, the Chinese medicine composition is prepared into a clinically acceptable Chinese medicine preparation according to conventional Chinese medicine preparation methods.

[0010] Furthermore, the Chinese medicine preparation is a granule, tablet, capsule, pill, powder, oral liquid, oral tablet, injection, gel, ointment, cream, solution, lotion, liniment, tincture or spray.

[0011] Furthermore, the Chinese medicine composition is used in the preparation of Chinese medicine preparations for brain protection, improving neuronal cell activity, and preventing, maintaining health or treating ischemic stroke.

[0012] According to another aspect of the present application, a method for screening a Chinese medicine composition for preventing and treating ischemic stroke is provided, characterized in that it comprises:

[0013] Step 1: The extracted active ingredients are mixed in a design to form test points with different composition ratios;

[0014] Step 2: Conducting cell experiments to detect changes in brain protection-related indicators in cells at each test point, wherein the brain protection-related indicators include nerve cell viability, lactate dehydrogenase, and neuron-specific enolase;

[0015] Step 3: Taking the brain protection related index measured in step 2 as the dependent variable and the proportion of each effective ingredient corresponding to each test point as the independent variable, a regression response model is established, the items included in the regression response model are screened by stepwise regression method, and the unknown coefficients in the regression response model are estimated by least squares method;

[0016] Step 4: Apply goodness-of-fit test and calculate R 2 and adjusted R 2Evaluate the fitted regression model;

[0017] Step 5: Perform multi-objective simultaneous optimization on the regression response model to obtain the optimal ratio of 3-caffeoylquinic acid, 4,5-dicaffeoylquinic acid and scutellarin.

[0018] Furthermore, step 6: experimentally verify the optimal ratio obtained in step 5 and evaluate the prediction deviation of the obtained model.

[0019] Furthermore, the type of the mixture design in step 1 is a simplex centroid design, a simplex grid point design or an extreme vertex design.

[0020] Furthermore, the regression response model is a quadratic model, a cubic model or a quartic model.

[0021] The beneficial effects of this application include:

[0022] The present application provides a Chinese medicine composition for preventing and treating ischemic stroke, in which 3-caffeoylquinic acid, 4,5-dicaffeoylquinic acid and scutellarin are scientifically matched, and the mutual compatibility has a synergistic effect to protect damaged neurons, improve nerve cell activity, and effectively reduce ischemic stroke damage. At the same time, the screening method of the composition uses a mixture design to establish a regression model with brain protection-related indicators as dependent variables and the proportion of each component as an independent variable, and the predictive ability of the model is evaluated. After the predictive ability of the model meets the standard, multiple brain protection indicators are simultaneously optimized to obtain the optimal ratio of each component and experimentally verify it. The Chinese medicine composition obtained by screening by this method has stable activity and definite effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of designing raw material test points using simplex centroid in one embodiment of the present application;

[0024] Figure 2 This is a schematic diagram showing the effects of different concentrations of Chinese medicine compositions on cell viability, lactate dehydrogenase and neuron-specific enolase levels in one embodiment of the present application;

[0025] Figure 3 This is a schematic diagram of the effects of different control groups on the neurological function of ischemic stroke mice in one embodiment of the present application;

[0026] Figure 4 This is a schematic diagram of the effects of different control groups on brain water content and cerebral infarction volume in ischemic stroke mice in one embodiment of the present application;

[0027] Figure 5 This is a schematic diagram of the effects of different control groups on the pathological morphology of brain tissue in mice with ischemic stroke in one embodiment of the present application;

[0028] Figure 6 This is a schematic diagram of the effects of different control groups on the neuronal morphology of brain tissue in ischemic stroke mice in one embodiment of the present application;

[0029] Figure 7 This is the effect of different control groups on serum lactate dehydrogenase and neuron-specific enolase in mice with ischemic stroke in one embodiment of the present application. DETAILED DESCRIPTION

[0030] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0031] Unless otherwise specified, the raw materials in the examples of this application were purchased through commercial channels.

[0032] Example 1: A Chinese medicine composition for preventing and treating ischemic stroke, characterized in that the effective ingredients of the Chinese medicine composition include the following substances in parts by weight: 30-50 parts of 3-caffeoylquinic acid, 20-40 parts of 4,5-dicaffeoylquinic acid and 30-50 parts of scutellarin.

[0033] Specifically, 3-caffeoylquinic acid is a type of caffeoyl component, which usually has pharmacological activities such as scavenging free radicals, anti-inflammatory, immunomodulatory and anticoagulant. 4,5-dicaffeoylquinic acid also belongs to the caffeoyl component and has significant anti-inflammatory activity. Studies have shown that its anti-inflammatory mechanism is related to inhibiting the migration of activated macrophages and reducing the release of O2. In addition, the series of phenolic acid compounds in Erigeron breviscapus not only have antioxidant activity, but also have the effects of dilating blood vessels and inhibiting thrombosis in the body. Scutellaria baicalensis is the main active ingredient in Erigeron breviscapus and belongs to the flavonoids. It is clinically used to treat cardiovascular and cerebrovascular diseases with good efficacy. It can improve the local blood circulation of cerebral vessels and has a positive effect on the treatment of ischemic cerebrovascular diseases.

[0034] The present application uses a scientific ratio of 3-caffeoylquinic acid, 4,5-dicaffeoylquinic acid and scutellarin in the composition, and their mutual compatibility has a synergistic effect to protect damaged neurons, increase the activity of nerve cells, and effectively reduce ischemic stroke damage.

[0035] Preferably, the effective ingredients of the traditional Chinese medicine composition include the following substances in parts by weight: 33 parts of 3-caffeoylquinic acid, 28 parts of 4,5-dicaffeoylquinic acid, and 39 parts of scutellarin;

[0036] Preferably, the active ingredient is derived from Erigeron breviscapus.

[0037] The Chinese medicine composition is prepared into a clinically acceptable Chinese medicine preparation according to a conventional Chinese medicine preparation method.

[0038] The Chinese medicine preparation is a granule, tablet, capsule, pill, powder, oral liquid, oral tablet, injection, gel, ointment, cream, solution, lotion, liniment, tincture or spray.

[0039] Specifically, granules: After the raw material drug is evenly mixed with suitable excipients, a granulation process is used to make a granular preparation, which is convenient for patients to take.

[0040] Tablets: After the raw material drug is mixed with the excipients, they are made into tablets through a tableting process, which is easy for patients to swallow and the dosage is accurate.

[0041] Capsules: Made by filling the raw materials into empty capsule shells, this can mask the bad smell of the drugs and improve patients’ compliance with medication.

[0042] Pills: After the raw material drug is mixed with the excipients, it is made into a pill-like preparation through a pill-making process. It is suitable for patients who need to take it for a long time.

[0043] Powder: The raw drug is crushed into fine powder and directly provided to patients for oral or external use. It has the characteristics of simple preparation and rapid effect.

[0044] Oral solution: The raw material drug is dissolved or dispersed in a suitable solvent to make a liquid preparation for patients to take orally, which is convenient for patients to take and absorb.

[0045] Oral tablets: such as buccal tablets and sublingual tablets, the raw materials are made into suitable shapes and sizes for patients to place in the mouth for slow dissolution or absorption. They are suitable for the treatment of oral diseases or systemic diseases that require rapid effects.

[0046] Injection: The raw material drug is dissolved or dispersed in a suitable solvent to make a sterile preparation for injection, which is suitable for the treatment of diseases that require rapid achievement of effective concentrations.

[0047] Gel: A semisolid preparation made by mixing the raw drug with the gel matrix evenly, suitable for external or local administration.

[0048] Ointments, creams and creams: All are suitable for external use. They are made by mixing the raw drug with a suitable matrix and have moisturizing, protective and therapeutic effects.

[0049] Solutions, lotions, liniments, tinctures and sprays: all are suitable for external use. The raw drug is dissolved or dispersed in a suitable solvent to make a preparation for the patient to apply, spray or wash.

[0050] It is worth noting that when preparing preparations, it is necessary to follow the preparation principles of pharmaceutical preparations, ensure the purity and stability of raw materials, and select appropriate excipients and preparation processes to meet the quality requirements of preparations and the medication needs of patients. In addition, necessary quality control and quality evaluation are required to ensure the safety and effectiveness of preparations. At the same time, specific preparation methods and quality control requirements should be formulated and implemented according to actual conditions and drug registration regulations.

[0051] In another aspect of the present application, the Chinese medicine composition is used in the preparation of Chinese medicine preparations for brain protection, improving neuronal cell activity, and preventing, maintaining health or treating ischemic stroke.

[0052] Embodiment 2: A method for screening a Chinese medicine composition for preventing and treating ischemic stroke, characterized in that it comprises:

[0053] Step 1: The extracted active ingredients are mixed in a design to form test points with different composition ratios;

[0054] Specifically, 3-caffeoylquinic acid, 4,5-dicaffeoylquinic acid and scutellarin are used as active ingredients and are mixed in different volume ratios to prepare a 1 ml mixture.

[0055] In this example, an enhanced simplex centroid mixture design is used, and the percentage of each component in the mixture ranges from 0 to 100%. A total of 10 test points are formed, and one test point refers to a unique component composition ratio, such as Figure 1 As shown, the specific composition ratios corresponding to the 10 test points are shown in Table 1:

[0056] Test point 3-Caffeoylquinic acid 4,5-dicaffeoylquinic acid Scutellarin 1 1.0 0.0 0.0 2 0.0 1.0 0.0 3 0.0 0.0 1.0 4 0.5 0.5 0.0 5 0.5 0.0 0.5 6 0.0 0.5 0.5 7 0.3333 0.3333 0.3333 8 0.6667 0.1667 0.1667 9 0.1667 0.6667 0.1667 10 0.1667 0.1667 0.6667

[0057] Table 1

[0058] Step 2: Conducting cell experiments to detect changes in brain protection-related indicators in cells at each test point, wherein the brain protection-related indicators include nerve cell viability, lactate dehydrogenase, and neuron-specific enolase;

[0059] Specifically, the experiment was conducted with mouse hippocampal neuron HT22 cells, cell culture was performed according to conventional methods, and the neuron oxygen-glucose deprivation / reperfusion model was replicated, and the different ratio compositions formed in step 1 were administered to the cells, and the cell viability was detected by CCK-8 method, and lactate dehydrogenase and neuron-specific enolase were detected by ELISA method. Among them, CCK-8 method and ELISA method are the most commonly used detection methods at present, and both methods are simple, rapid, sensitive and accurate, and both have mature commercial reagents.

[0060] In this embodiment, each test point was independently repeated 3 times, and the results were expressed as "mean ± standard deviation", as shown in Table 2:

[0061]

[0062] Table 2

[0063] Step 3: Taking the brain protection related index measured in step 2 as the dependent variable and the proportion of each effective ingredient corresponding to each test point as the independent variable, a regression response model is established, wherein the items included in the regression response model are screened by the stepwise regression method, and the unknown coefficients in the regression response model are estimated by the least squares method;

[0064] Specifically, in this embodiment, in order to establish a regression response model, and use the stepwise regression method to screen variables and the least squares method to estimate coefficients, the following method can be used:

[0065] Collect data:

[0066] Take the brain protection related indicators measured in step 2 (e.g., the values ​​of nerve cell viability, lactate dehydrogenase, and neuron-specific enolase) as the dependent variable, and the proportion of each raw material corresponding to each test point as the independent variable. Arrange the dependent and independent variables into a data format suitable for regression analysis. Usually, this includes one dependent variable column and multiple independent variable columns.

[0067] Set the initial model:

[0068] The initial model can include all independent variables.

[0069] Further, the stepwise regression method was used to screen variables:

[0070] Stepwise regression methods include forward selection, backward elimination, and stepwise selection (a combination of the first two). Here, we can choose the stepwise selection method.

[0071] Set the parameters of stepwise regression in the statistical software and perform the stepwise regression process.

[0072] The software will automatically calculate the value based on a criterion (such as AIC, BIC, or adjusted R 2 ) to select or exclude variables.

[0073] Record the final selected variables and corresponding coefficients.

[0074] Establish the final regression model: write the final regression equation based on the screened variables and coefficients.

[0075] Evaluate the model:

[0076] Use statistical indicators (such as R 2 , adjusted R 2etc.) to evaluate the model's goodness of fit and predictive ability.

[0077] Perform residual analysis to check whether the model meets the assumptions of linear regression.

[0078] Interpret and apply the model: Explain the meaning of each coefficient in the model and their impact on the dependent variable. Use the model to make predictions or optimizations. For example, the ratio of raw materials can be adjusted to predict changes in indicators related to brain protection.

[0079] It is worth noting that in one embodiment of the present application, a quartic polynomial model was used, and regression equations for the response indicators cell viability, lactate dehydrogenase, and neuron-specific enolase were obtained through stepwise regression analysis. The P values ​​obtained from the global F test of each model were all less than 0.05, indicating that each model had significant statistical significance.

[0080] Step 4: Apply goodness-of-fit test and calculate R 2 (coefficient of determination) and adjusted R 2 (calibrated determination coefficient) to evaluate the fitted regression model;

[0081] Specifically, the goodness-of-fit test showed that R 2 With adjusted R 2 are both large, close to 1, and adjusted R 2 With R 2 There is no obvious attenuation compared to the control, indicating that each model fits well and has good predictive ability. The results are shown in Table 3:

[0082]

[0083] Table 3

[0084] Step 5: Perform multi-objective synchronous optimization on the regression response model, wherein the multi-objective synchronous optimization can be realized by existing software. Obtain the optimal ratio of 3-caffeoylquinic acid, 4,5-dicaffeoylquinic acid and scutellarin;

[0085] Specifically, taking cell viability, lactate dehydrogenase and neuron-specific enolase as response indicators at the same time, an optimal ratio composition was obtained, which included the following raw materials in parts by weight: 33 parts of 3-caffeoylquinic acid, 28 parts of 4,5-dicaffeoylquinic acid, and 39 parts of scutellarin.

[0086] Step 6: Experimentally verify the optimal ratio obtained in step 5 and evaluate the prediction deviation of the obtained model.

[0087] Specifically, according to the deviation calculation formula "|predicted value-measured value| / measured value×100%", the measured values ​​and predicted values ​​of cell viability and neuron-specific enolase are relatively close, indicating that the optimal ratio and predicted response value obtained by the model prediction have a certain degree of reliability. Although the model has a certain deviation in predicting the lactate dehydrogenase level, it still guides us to find a ratio composition scheme that is not inferior to that of Erigeron breviscapus in effectively reducing the lactate dehydrogenase level. As shown in Table 4:

[0088]

[0089] Table 4

[0090] It is worth noting that GraphPadPrism 8 software was used to compare the mean of each experimental group and draw statistical graphs. One-way ANOVA was used to perform an overall comparison of the mean between each experimental group, and then the Holm-Sidak test was used to perform pairwise comparisons between groups. Among them, GraphPad Prism 8 is a powerful data analysis software that provides convenient and easy-to-use statistical analysis and drawing functions, including one-way ANOVA and Holm-Sidak test. One-way ANOVA is a commonly used statistical method for comparing the mean differences of three or more independent samples. In this application, it can be used to compare whether there are significant differences in the means between each experimental group. If the results of ANOVA show that there are significant differences in the means of at least two groups, then further pairwise comparisons between groups are required to determine which groups have significant differences. The Holm-Sidak test is a statistical method for multiple comparisons, which can be used to compare whether the differences between two groups are significant after analysis of variance. The Holm-Sidak test performs well in controlling the false positive rate and is therefore widely used in practical applications.

[0091] In summary, GraphPad Prism 8 software was used to compare the means of the experimental groups and draw statistical graphs, One-way ANOVA was used for overall comparison, and then the Holm-Sidak test was used for pairwise comparison. This process can more accurately understand the mean differences between the experimental groups and provide valuable guidance for subsequent experimental design and data analysis.

[0092] The results showed that compared with oxygen-glucose deprivation / reperfusion model cells, the Chinese medicine composition of the present application significantly increased the viability of damaged neuronal cells (P<0.01), and significantly restored the levels of lactate dehydrogenase (P<0.01) and neuron-specific enolase (P<0.05) in damaged neuronal cells. Among them, oxygen-glucose deprivation / reperfusion (OGD / R) model cells are a commonly used in vitro research model, especially in the study of ischemic stroke. The effects of different concentrations of Chinese medicine compositions on cell viability, lactate dehydrogenase and neuron-specific enolase levels are shown in the figure. Figure 2 As shown, Figure 2 (a) is the effect of the Chinese medicine composition on the activity of brain cells in ischemic stroke mice, Figure 2 (b) is the effect of the Chinese medicine composition on brain lactate dehydrogenase in mice with ischemic stroke, Figure 2 (c) is the effect of the Chinese medicine composition on neuron-specific enolase in ischemic stroke mice.

[0093] The type of the mixture design in step 1 is a simplex centroid design, a simplex grid point design or an extreme vertex design.

[0094] Specifically, the simplex centroid design ensures adequate coverage of the area of ​​interest on the response surface. It constructs a response surface model by selecting a set of experimental points that are located at the centroid (i.e., center point) of the simplex and at the vertices or edges of the simplex. This approach is very useful when you need to fully explore the response surface and consider the effects of multiple component proportions.

[0095] Simplex grid design is a design method based on simplex grid points. It constructs the model by selecting experimental points at each vertex of the simplex and at the grid points. This method is suitable for situations where you need to explore the response surface more finely and consider the interaction between the component proportions.

[0096] Extreme vertex design is a design method used in mixture design when the components are often subject to upper and lower limits. It constructs the model by selecting the extreme vertices located at the intersection of each restriction plane as the experimental points. This method is particularly suitable for situations where the proportions of the components are strictly restricted, and can more accurately reflect the impact of these restrictions on the response variable.

[0097] In one embodiment of the present application, a simplex centroid design is adopted, and the schematic diagram of the test points after the design is as follows: Figure 1 shown.

[0098] The regression response model is a quadratic model, a cubic model or a quartic model.

[0099] Specifically, the quadratic model is usually expressed as Y = a + bX + cX 2 (where a, b, c are coefficients, X is the independent variable, and Y is the dependent variable). It contains a linear term and a square term, which can describe the parabolic relationship between the independent variable and the dependent variable. It is often used to describe phenomena that increase with the increase of the independent variable within a certain range, but decrease with the increase of the independent variable after exceeding a certain point.

[0100] The cubic model is usually expressed as Y = a + bX + cX 2 +dX 3 (where a, b, c, d are coefficients). It contains a linear term, a square term, and a cubic term, and can describe more complex nonlinear relationships, including possible inflection points or extreme points. It is suitable for situations where it is necessary to describe more complex nonlinear relationships between independent variables and dependent variables.

[0101] The quartic model is usually expressed as Y = a + bX + cX 2 +dX 3 +eX 4 (where a, b, c, d, e are coefficients). It contains a linear term, a square term, a cubic term, and a quartic term, and can describe more complex nonlinear relationships, including multiple inflection points or extreme points. When you need to capture very complex nonlinear relationships between independent variables and dependent variables, the quartic model may be a suitable choice.

[0102] In this application, a quartic model is selected, and for the response indicators of cell viability, lactate dehydrogenase and neuron-specific enolase, we can establish a quartic polynomial regression equation for them respectively. These equations will contain linear, quadratic, cubic and quartic terms (as well as possible interaction terms and constant terms) of the independent variables (such as raw material ratio, processing time, temperature, etc.) related to each response indicator, but which specific terms are included in the final equation will depend on the results of the stepwise regression process. This application forms corresponding indicators by using cell viability, lactate dehydrogenase and neuron-specific enolase as independent variables.

[0103] Example 3: Example 1 selected from Example 2 was used to treat ischemic stroke mice. SPF male C57BL / 6 mice were used to replicate the middle cerebral artery occlusion model according to conventional methods. Example 1 was administered to ischemic stroke mice, and the neurological function of the mice was evaluated using the Longa score, mNSS score, corner turning test, sticky stick removal test, and cylinder test. The Longa score evaluates the limb movement and conscious cognitive state of the mice, the mNSS score comprehensively evaluates the reflex function, sensory function, and motor coordination ability of the mice, the corner turning test evaluates the sensory and motor function of the mice, the sticky stick removal test comprehensively evaluates the motor coordination and degree of dystonia of the mice, and the cylinder test evaluates the degree of asymmetry of the limbs of the mice. The above five methods are relatively common, simple to operate, and rapid methods for evaluating mouse behavior. There were 8 animals in each group. The results showed that the Chinese medicine composition provided in this application can improve the neurological function of ischemic stroke mice (P<0.01), promote the recovery of their motor function and autonomous activity, and reduce cerebral ischemia / reperfusion injury. The effects on the neurological function of ischemic stroke mice under different control groups are shown in the figure. Figure 3 As shown, Figure 3 (a) Evaluation of the effects of different control groups on limb movement and consciousness and cognitive status of ischemic stroke mice. Figure 3 (b) Evaluation of the effects of different control groups on the reflex function, sensory function and motor coordination ability of ischemic stroke mice. Figure 3 (c) Evaluation of the effects of different control groups on the sensory and motor functions of ischemic stroke mice. Figure 3 (d) is the evaluation of the effects of different control groups on motor coordination and dystonia in ischemic stroke mice. Figure 3 (e) is the evaluation of the effect of different control groups on the asymmetry of the limbs of ischemic stroke mice. Figure 3 The different control groups in the middle horizontal axis are the normal group, the oxygen glucose deprivation / reperfusion model group, the Chinese medicine composition group in Example 1 of the present application (specifically, the Chinese medicine composition obtained by mixing 33 parts of 3-caffeoylquinic acid, 28 parts of 4,5-dicaffeoylquinic acid, and 39 parts of scutellarin from Erigeron breviscapus and Erigeron breviscapus group);

[0104] It is worth noting that the Erigeron breviscapus group mentioned in the present application is all the Chinese patent medicine Erigeron breviscapus granules, which are composed of 75% ethanol extract of Erigeron breviscapus.

[0105] The degree of brain tissue edema was determined by detecting brain water content, and the degree of brain tissue infarction was evaluated by TTC staining. There were 3 animals in each group. The results showed that the Chinese medicine composition provided by the present application can significantly reduce brain water content (P<0.01) and cerebral infarction volume (P<0.01). The effects of different control groups on brain water content and cerebral infarction volume in ischemic stroke mice are shown in Figure 2. Figure 4 As shown, Figure 4 (a) Schematic diagram of the effect of different control groups on brain water content in ischemic stroke mice; Figure 4 (b) is a schematic diagram showing the effect of different control groups on the cerebral infarction volume in ischemic stroke mice. Figure 4 (c) Effects of different control groups on brain water content and cerebral infarction in mice with ischemic stroke. Figure 4 The different control groups in the horizontal axis in (a) and (b) are the normal group, the oxygen-glucose deprivation / reperfusion model group, the Chinese medicine composition group in Example 1 of the present application (specifically, a Chinese medicine composition derived from Erigeron breviscapus and obtained by mixing 33 parts of 3-caffeoylquinic acid, 28 parts of 4,5-dicaffeoylquinic acid, and 39 parts of scutellariae) and the Erigeron breviscapus group.

[0106] Brain tissue morphology can be detected by HE staining to determine the pathological condition of brain tissue. The results showed that compared with the normal group, a large number of nerve cells in the model group were sparsely arranged, with irregular outlines and cell shrinkage. Cracks and vacuoles caused by cell shrinkage were visible around the cells. The chromatin of the cell nuclei was dense and the staining was significantly deepened. Compared with the model group, the cell structure of the Chinese medicine combination group and the Erigeron breviscapus group tended to be normal, the cell outlines were more regular, the degree of cell shrinkage, surrounding cracks and vacuoles were reduced to varying degrees, and the staining of the cell nucleus became lighter, indicating that different concentrations of Chinese medicine combination can improve the morphology of brain tissue cells. Effects of different control groups on the pathological morphology of brain tissue in mice with ischemic stroke are shown in the figure. Figure 5 It is worth noting that Figure 5 The green arrows in the middle indicate the cracks and vacuoles around the cells due to cell shrinkage; the red arrows indicate that the chromatin of the cell nucleus is dense and the staining is significantly deepened; Scale bar: 50μm.

[0107] The ultrastructure of brain tissue neurons was observed using transmission electron microscopy. The results showed that compared with the normal group, the neuronal cell morphology of the model group was irregular, the nuclear membrane was broken and blurred, and the mitochondrial cristae were broken and vacuolated; compared with the model group, the neuronal morphology of the Chinese medicine combination group and the Erigeron breviscapus group was significantly improved, the nuclear membrane and mitochondrial structure were basically intact, and the cristae were slightly damaged. Effects of different control groups on the morphology of brain tissue neurons in mice with ischemic stroke Figure 6 As shown, it is worth noting that the green arrow indicates nuclear membrane rupture, and the red arrow indicates mitochondrial cristae breakage and vacuolization; Scale bar: 10 μm.

[0108] Serum lactate dehydrogenase and neuron-specific enolase were detected by ELISA. ELISA is simple, rapid, sensitive, accurate, and has mature commercial reagents. There were 3 animals in each group. The results showed that compared with the model group, the lactate dehydrogenase (P<0.01) and neuron-specific enolase (P<0.05) levels in the Chinese medicine combination group were significantly reduced. Effects of different control groups on serum lactate dehydrogenase and neuron-specific enolase in mice with ischemic stroke Figure 7 As shown, Figure 7 (a) Schematic diagram of the effect of different control groups on serum lactate dehydrogenase in mice with ischemic stroke;

[0109] Figure 7 (b) is a schematic diagram showing the effects of different control groups on neuron-specific enolase in ischemic stroke mice. Figure 7 The different control groups in the middle horizontal axis are the normal group, the oxygen-glucose deprivation / reperfusion model group, the Chinese medicine composition group in Example 1 of the present application (specifically a Chinese medicine composition derived from Erigeron breviscapus and obtained by mixing 33 parts of 3-caffeoylquinic acid, 28 parts of 4,5-dicaffeoylquinic acid, and 39 parts of scutellariae) and the Erigeron breviscapus group.

[0110] The above test results show that the Chinese medicine composition in the present application has a significant improvement effect on ischemic stroke, can protect damaged neurons, increase nerve cell activity, and effectively reduce ischemic stroke damage. Among them, Example 1 is the best composition, and Example 2 is the best screening method. The improvement effect on ischemic stroke is statistically significant compared with the brain injury group.

[0111] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A Chinese medicine composition for preventing and treating ischemic stroke, characterized in that: The effective ingredients of the traditional Chinese medicine composition include the following substances in parts by weight: 30-50 parts of 3-caffeoylquinic acid, 20-40 parts of 4,5-dicaffeoylquinic acid and 30-50 parts of scutellarin.

2. A Chinese medicine composition for preventing and treating ischemic stroke according to claim 1, characterized in that: The effective ingredients of the traditional Chinese medicine composition include the following substances by weight: 33 parts of 3-caffeoylquinic acid, 28 parts of 4,5-dicaffeoylquinic acid, and 39 parts of scutellarin; Preferably, the active ingredient is derived from Erigeron breviscapus.

3. The Chinese medicine composition for preventing and treating ischemic stroke according to claim 1, characterized in that: The Chinese medicine composition is prepared into a clinically acceptable Chinese medicine preparation according to a conventional Chinese medicine preparation method.

4. A Chinese medicine composition for preventing and treating ischemic stroke according to claim 3, characterized in that: The Chinese medicine preparation is a granule, tablet, capsule, pill, powder, oral liquid, oral tablet, injection, gel, ointment, cream, solution, lotion, liniment, tincture or spray.

5. A Chinese medicine composition for preventing and treating ischemic stroke according to any one of claims 1 to 4, characterized in that: The Chinese medicine composition is used in preparing Chinese medicine preparations for brain protection, improving neuronal cell activity, and preventing, maintaining health or treating ischemic stroke.

6. A method for screening a Chinese medicine composition for preventing and treating ischemic stroke, characterized in that: include: Step 1: The extracted active ingredients are mixed in a design to form test points with different composition ratios; Step 2: Conducting a cell experiment to detect changes in brain protection-related indicators in cells at each test point, wherein the brain protection-related indicators include nerve cell viability, lactate dehydrogenase, and neuron-specific enolase; Step 3: Taking the brain protection related index measured in step 2 as the dependent variable and the proportion of each effective ingredient corresponding to each test point as the independent variable, a regression response model is established, the items included in the regression response model are screened by stepwise regression method, and the unknown coefficients in the regression response model are estimated by least squares method; Step 4: Apply goodness-of-fit test and calculate R 2 and adjusted R 2 Evaluate the fitted regression model; Step 5: Perform multi-objective simultaneous optimization on the regression response model to obtain the optimal ratio of 3-caffeoylquinic acid, 4,5-dicaffeoylquinic acid and scutellarin.

7. The method for screening a Chinese medicine composition for preventing and treating ischemic stroke according to claim 6, characterized in that: The method further comprises: Step 6: Experimentally verify the optimal ratio obtained in step 5 and evaluate the prediction deviation of the obtained model.

8. The method for screening a Chinese medicine composition for preventing and treating ischemic stroke according to claim 6, characterized in that: The type of the mixture design in step 1 is a simplex centroid design, a simplex grid point design or an extreme vertex design.

9. The method for screening a Chinese medicine composition for preventing and treating ischemic stroke according to claim 6, characterized in that: The regression response model is a quadratic model, a cubic model or a quartic model.

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