Traditional Chinese medicine active ingredient composition for treating cerebral infarction and application thereof

Abstract

The invention discloses a traditional Chinese medicine active ingredient composition for treating cerebral infarction and application thereof, and relates to the technical field of traditional Chinese medicines. By optimizing the dosage ratio of the active ingredient compound (calycosin-7-glucoside, formononetin and astragaloside), it is proved that the compound remarkably relieves neurological impairment, reduces the cerebral infarction area, promotes neurogenesis after cerebral infarction and can effectively improve cerebral infarction prognosis, and the effect is equivalent to that of a clinical proved recipe 'Yang-tonifying and five-returning decoction'. The active ingredient composition obtained by the invention has the advantages of clear ingredients, definite mechanism and controllable quality, the safety of clinical medication is improved on the basis of ensuring the curative effect, the hidden danger of adverse reaction is reduced, and a modern compound traditional Chinese medicine which is compatible with active ingredients in a modern dosage form is favorably prepared.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and particularly relates to a composition of active ingredients of traditional Chinese medicine for treating cerebral infarction and its application. Background Art

[0002] At present, stroke has become the second leading cause of death and the third leading cause of disability globally, seriously affecting the quality of life of patients and leading to an increasing economic burden on healthcare. Clinically, stroke can be divided into ischemic stroke and hemorrhagic stroke, among which about 80% are ischemic strokes, also known as cerebral infarction. The pathophysiological mechanism of ischemic stroke is complex. In the acute phase, it is mainly due to hypoxia-ischemia of brain tissue, resulting in intracellular calcium overload, disordered release of neurotransmitters, apoptosis and neuronal necrosis, etc.; while in the subacute and chronic phases, it is mainly focused on nerve repair and functional reconstruction. Intravenous thrombolysis and endovascular treatment are the two primary strategies for the treatment of acute cerebral infarction clinically. However, there are unfavorable factors such as a narrow treatment time window, hemorrhagic transformation and reperfusion injury, and the number of clinically benefited patients is limited. In addition, due to the poor hypoxia tolerance of nerve cells, even if the blood vessels are recanalized, the death of neurons in the infarct core area is inevitable, resulting in sequelae in patients. Due to the very complex pathophysiological mechanism of ischemic brain injury, most of the clinical trials of neuroprotective drugs developed for ischemic brain injury have ended in failure so far. However, nerve repair can not only promote the reconstruction of nerve function after stroke, but also has a longer treatment time window. Therefore, exploring drugs that can promote nerve repair in cerebral infarction is of great clinical significance.

[0003] Neurogenesis plays an important role in nerve repair in the subacute and chronic phases after cerebral infarction and has received increasing attention. A number of studies have shown that regulating endogenous neurogenesis can improve nerve function repair after stroke, inhibiting neurogenesis can exacerbate nerve function deficits after stroke and hinder recovery, and nerve function can be improved by transplanting neural progenitor cells. However, the biological process of neurogenesis is complex, which involves multiple links such as the proliferation, migration, differentiation, and axonal regeneration of neural stem cells (NSCs). Therefore, it is impossible to completely repair nerve function after stroke only through single-target intervention.

[0004] Traditional Chinese medicine holds that qi deficiency and blood stasis are the main pathogenesis of cerebral infarction, and the treatment mainly focuses on supplementing qi, activating blood circulation, and dredging collaterals. Buyang Huanwu Decoction (BYHWD) is a representative prescription for treating cerebral infarction in traditional Chinese medicine, which is composed of Astragalus membranaceus, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, Carthamus tinctorius, Prunus persica, and Pheretima aspergillum. The compatibility feature is the combination of a large dose of qi-supplementing herb Astragalus membranaceus and a small dose of herbs for activating blood circulation and removing stasis, enabling qi to promote blood circulation, activating blood circulation without damaging healthy qi, and jointly achieving the effects of supplementing qi, activating blood circulation, and dredging collaterals. Previous studies in our laboratory found that BYHWD promoted the long-term recovery of sensory, motor, and learning and memory functions in rats with cerebral ischemia (Chu Lisheng, Yang Wuming, Shao Liang, et al. Effects of Buyang Huanwu Decoction on learning and memory functions in rats with focal cerebral ischemia. Chinese Journal of Behavioral Medicine Science 2005;14(12):1066 - 1067. Chu Lisheng, Shao Liang, Meng Dingyu, et al. Long-term protective effects of Buyang Huanwu Decoction on focal cerebral ischemia injury in rats. Chinese Journal of Clinical Rehabilitation 2006;10(11):56 - 58.). Further studies found that BYHWD promoted angiogenesis, neurogenesis, axon regeneration, and synaptic plasticity after cerebral ischemia (Chu Lisheng, Jiang Yanyan, Ke Qing, et al. Buyang Huanwu Decoction promotes angiogenesis and functional recovery after focal cerebral ischemia in rats. Chinese Archives of Traditional Chinese Medicine 2011;29(2):335 - 337. Chu Lisheng, Yin Yuanjun, Ke Qing, et al. Effects of Buyang Huanwu Decoction on angiogenesis and Ang-1 / Tie-2 expression in focal cerebral ischemia mice. Chinese Journal of Behavioral Medicine and Brain Science 2011,20(3):202 - 204. Chu Lisheng, Yu Tianhong, Liu Zhiting, et al. Effects of Buyang Huanwu Decoction on the proliferation and survival of neural stem cells in the dentate gyrus of the hippocampus after focal cerebral ischemia in rats. Journal of Zhejiang Chinese Medical University 2011,35(3):375 - 377. Liu Zhiting, Yu Tianhong, Qu Tiebing, et al. Effects of Buyang Huanwu Decoction and its disassembled formulas on growth-associated protein 43 and synaptophysin expression after focal cerebral ischemia in mice. Chinese Journal of Behavioral Medicine and Brain Science, 2012,21(12):1070 - 1072. Li Lin, Liu Zhiting, Chu Lisheng, et al. Buyang Huanwu Decoction induces angiogenesis after cerebral ischemia and promotes the migration of neuroblasts in the subventricular zone. China Journal of Chinese Materia Medica, 2015;40(2):298 - 302.). Through the study of disassembled formulas, it was found that BYHWD promoting neurogenesis after cerebral infarction was mainly closely related to the large dose of qi-supplementing herb Astragalus membranaceus (Yu Tianhong, Chu Lisheng, Liu Zhiting, et al. Effects of different doses of Buyang Huanwu Decoction on the proliferation of neural stem cells after cerebral ischemia in rats. Chinese Journal of Experimental Traditional Medical Formulae, 2013,19(7):182 - 185. Qu Tiebing, Yu Tianhong, Liu Zhiting, et al. Effects of Buyang Huanwu Decoction and its disassembled formulas on neurogenesis after cerebral ischemia in rats. Chinese Journal of Integrated Traditional and Western Medicine, 2014,34(3):342 - 347.). Therefore, Buyang Huanwu Decoction exerts its pharmacological effects through multiple components, multiple targets, and multiple pathways through its compatibility combination, and has obvious advantages compared with drugs with a single component and a single target.However, the mechanism of action of traditional Chinese herbal medicines is difficult to clarify due to their numerous chemical components. At the same time, the origin of the medicinal materials, storage and maintenance conditions, and processing methods may lead to different therapeutic effects, limiting their clinical application and promotion as well as their international and modern development.

[0005] In order to better play the advantages of traditional Chinese medicine, it is necessary to conduct in-depth research on the pharmacological substances and action mechanisms of prescription compatibility. The effective ingredient compatibility of traditional Chinese medicine is based on the theory of compatibility combined with modern technology to identify the effective ingredients, confirm the pharmacological functions, optimize the dosage and study the mechanism to achieve the purpose of reducing toxicity and increasing efficacy. It is derived from traditional Chinese medicine, with accurate pharmacological substances and controllable quality. It is also suitable for studying its mechanism of action at the animal and cell levels. It not only has the advanced nature of modern pharmaceutical manufacturing, but also retains the traditional characteristics of traditional Chinese medicine compatibility, providing new ideas for the innovative development of traditional Chinese medicine. Summary of the invention

[0006] In view of this, the present invention discloses a Chinese medicinal active ingredient composition for treating cerebral infarction and its application. The Chinese medicinal active ingredient composition (calycosin, formononetin and astragaloside IV) in the present invention obtains the best compatible combination from the active ingredient combinations in different proportions, and has the characteristics of multi-component, multi-pathway and multi-effect effects in promoting nerve repair after cerebral infarction.

[0007] A series of pathophysiological reactions triggered by cerebral infarction, including inflammatory response, oxidative stress and blood-brain barrier damage, will aggravate brain tissue damage, but also initiate a certain degree of self-repair mechanism. In neural repair after cerebral infarction, endogenous neurogenesis is an important link. Endogenous neurogenesis refers to the proliferation, migration and differentiation of neural stem cells (NSCs) in the brain around the infarct. These new neurons and supporting cells can partially replace damaged cells and participate in the reconstruction of neural networks. However, these endogenous repair capabilities are usually not enough to completely restore damaged neural function. In short, neural repair after cerebral infarction is a complex multifactorial process, including neurogenesis, angiogenesis and inflammatory regulation. In-depth study of its mechanism and development of effective intervention methods are of great significance for improving the prognosis of patients with cerebral infarction.

[0008] The invention provides a traditional Chinese medicine active ingredient composition for treating cerebral infarction. The effective ingredients of the traditional Chinese medicine active ingredient composition include calycosin-7-glucoside (CLG), formononetin (FMNT) and astragaloside IV (AS-IV).

[0009] Calycosin-7-O-β-D-glucoside is a flavonoid compound, which is mostly found in leguminous plants and is one of the quality control index components of Astragalus membranaceus. It has antioxidant stress and anti-apoptosis activities and has a certain protective effect on nerve cells. The molecular formula of calycosin-7-O-β-D-glucoside is C 22 H 22 O 10 , CAS No.: 20633-67-4, and the chemical formula is as follows:

[0010]

[0011] Formononetin is a natural flavonoid compound that widely exists in various plants, especially leguminous plants such as Astragalus membranaceus, and has multiple pharmacological effects such as anti-inflammatory, antioxidant, and neuroprotective effects. The molecular formula of formononetin is C 16 H 12 O 4 , CAS No.: 485-72-3, and the chemical formula is as follows:

[0012]

[0013] Astragaloside IV is a natural saponin compound and the main active ingredient in Astragalus membranaceus. Due to its multiple pharmacological effects, it shows significant therapeutic potential in the fields of immunomodulation, anti-inflammatory, antioxidant, cardiovascular protection, and neuroprotection. The molecular formula of astragaloside IV is C 41 H 68 O 14 , CAS No.: 84687-43-4, and the chemical formula is as follows:

[0014]

[0015] Specifically, the dosage of calycosin-7-O-β-D-glucoside in the traditional Chinese medicine active ingredient composition is 1-33 mg / kg per day, the dosage of formononetin is 0.2-25.6 mg / kg per day, and the dosage of astragaloside IV is 1.25-100 mg / kg per day.

[0016] Further preferably, the dosage of calycosin-7-O-β-D-glucoside in the traditional Chinese medicine active ingredient composition is 21-33 mg / kg per day, the dosage of formononetin is 6.4-25.6 mg / kg per day, and the dosage of astragaloside IV is 20-60 mg / kg per day.

[0017] Most preferably, the dosage of calycosin-7-O-β-D-glucoside in the traditional Chinese medicine active ingredient composition is 32.8 mg / kg per day, the dosage of formononetin is 25.0 mg / kg per day, and the dosage of astragaloside IV is 34.1 mg / kg per day.

[0018] The present invention also provides the application of the traditional Chinese medicine active ingredient composition in the preparation of a drug for treating cerebral infarction.

[0019] In some embodiments of the present invention, the dosage form of the drug is a liquid preparation, a powder for injection, a tablet, a capsule, a powder, a pill, an oral liquid, an ointment, a granule or a dressing.

[0020] Preferably, when the dosage form of the drug is a liquid preparation, the administration method includes oral administration;

[0021] The dosage of calycosin-7-O-β-D-glucoside in the drug is 1-33 mg / kg per day, the dosage of formononetin is 0.2-25.6 mg / kg per day, and the dosage of astragaloside IV is 1.25-100 mg / kg per day.

[0022] More preferably, the dosage of calycosin-7-O-β-D-glucoside in the pharmaceutical composition is 21-33 mg / kg per day, the dosage of formononetin is 6.4-25.6 mg / kg per day, and the dosage of astragaloside IV is 20-60 mg / kg per day.

[0023] Most preferably, the dosage of calycosin-7-O-β-D-glucoside in the drug is 32.8 mg / kg per day, the dosage of formononetin is 25.0 mg / kg per day, and the dosage of astragaloside IV is 34.1 mg / kg per day.

[0024] The present invention is a compound pharmaceutical composition composed of three traditional Chinese medicine active ingredients, namely calycosin-7-O-β-D-glucoside, formononetin and astragaloside IV; the compound pharmaceutical composition of the present invention is based on the key technology of quantitative design of traditional Chinese medicine compound. Through uniform design experiments and mathematical models, the dosage ratio of the best pharmacodynamic effect is predicted. Finally, the pharmacodynamic effect is verified by animal experiments to obtain the best combination. This method is reasonable and feasible; the composition of the present invention is clear, the quality is controllable, while ensuring the curative effect, the dosage of taking medicine is reduced, the safety of clinical medication is improved, the hidden danger of adverse reactions is reduced, which is conducive to the preparation of modern compound traditional Chinese medicine with active ingredient compatibility in modern dosage forms, and has the characteristics of multi-component, multi-way and multi-effect treatment of cerebral infarction.

[0025] The beneficial effects of the present invention:

[0026] By optimizing the dosage ratio of the active ingredient compound, the present invention proves that this compound (especially CLG 32.8 mg / kg, FMNT 25.0 mg / kg, AS-IV 34.1 mg / kg, once a day) can significantly reduce neurological deficits, reduce the area of cerebral infarction, and promote neurogenesis after cerebral infarction, and can effectively improve the prognosis of cerebral infarction. The effect is equivalent to that of the clinical prescription "Buyang Huanwu Decoction". The active ingredient composition obtained by the present invention has the advantages of clear composition, clear mechanism and controllable quality. While ensuring the curative effect, the safety of clinical medication is improved, the hidden danger of adverse reactions is reduced, which is conducive to the preparation of modern compound traditional Chinese medicine with active ingredient compatibility in modern dosage forms. Description of the Drawings

[0027] Figure 1 To show the effects of each treatment in the uniform design experiment on the recovery of neurological function in rats after cerebral ischemia; among them, A is the line graph of the mNSS score results of rats; B is the results of the corner test of rats; Sham: sham operation group; MCAO / R: model group; Edaravone: edaravone group; CFA-1 to 9: uniform design groups 1 to 9.

[0028] Figure 2 To show the effects of each group in the uniform design verification experiment on the recovery of neurological function in rats after cerebral ischemia; among them, A is the mNSS score and B is the number of right turns.

[0029] Figure 3 To show the effects of each group of treatments on the infarct area; among them, A is the stained map of rat brain sections and B is the statistical graph of the infarct area; compared with the Sham group, **P < 0.01; compared with the MCAO / R group, ##P < 0.01; compared with the CFA-OP group, &P < 0.05.

[0030] Figure 4 To show that the effective active ingredient combination of BYHWD increases the number of BrdU + / Nestin + cells in rats with cerebral ischemia; among them, A is the immunofluorescence staining map of rat brain tissue sections; B is the number of BrdU + / Nestin + cells in rats with cerebral ischemia; compared with the Sham group, **P < 0.01; compared with the MCAO / R group, ##P < 0.01; compared with the CFA-6 group, &&P < 0.01; compared with the CFA-OP group, $$P < 0.01.

[0031] Figure 5 To show that the effective active ingredient combination of BYHWD increases the number of BrdU + / DCX + cells in rats with cerebral ischemia; among them, A is the immunofluorescence staining map of rat brain tissue sections; B is the number of BrdU + / DCX + cells in rats with cerebral ischemia; compared with the Sham group, *P < 0.05; compared with the MCAO / R group, ##P < 0.01; compared with the CFA-6 group, &&P < 0.01; compared with the CFA-OP group, $$P < 0.01.

[0032] Figure 6 To show that the effective active ingredient combination of BYHWD increases the number of BrdU + / NeuN + cells in rats with cerebral ischemia; among them, A is the immunofluorescence staining map of rat brain tissue sections; B is the number of BrdU+ / NeuN + Number of cells; compared with the Sham group, **P < 0.01; compared with the MCAO / R group, ##P < 0.01; compared with the CFA-6 group, &&P < 0.01; compared with the CFA-OP group, $$P < 0.01.

[0033] Figure 7 The effective active ingredient combination of BYHWD increases BrdU in rats with cerebral ischemia + / GFAP + Number of cells; among them, A is the immunofluorescence staining map of rat brain tissue sections; B is BrdU in rats with cerebral ischemia + / GFAP + Number of cells; compared with the Sham group, **P < 0.01; compared with the MCAO / R group, ##P < 0.01; compared with the CFA-OP group, $$P < 0.01, ns indicates P > 0.05.

[0034] Figure 8 CLG promotes the expression of p-EGFR in NSCs after OGD / R; among them, A is the effect of different concentrations of CLG on EGFR phosphorylation; B is the effect of CLG on EGFR phosphorylation after pretreatment with the EGFR tyrosine kinase inhibitor (AG-1478); compared with the Control group, **P < 0.01; compared with the OGD / R group, #P < 0.05, ##P < 0.01; compared with the CLG group, &&P < 0.01.

[0035] Figure 9 CLG promotes the proliferation of NSCs after OGD / R by upregulating the phosphorylation level of EGFR; among them, A is the immunofluorescence test result of CLG promoting the proliferation of NSCs after OGD / R; B is BrdU + / Nestin + Proportion change of cells; compared with Control, **P < 0.01; compared with the OGD / R group, ##P < 0.01; compared with the CLG group, &&P < 0.01.

[0036] Figure 10 FMNT promotes the expression of MMP-2 / 9 in NSCs after OGD / R; among them, A and B are the effects of different concentrations of FMNT on the expression of MMP-2 and MMP-9; C and D are the effects of FMNT on the expression of MMP-2 and MMP-9 after treatment with the MMP2 / 9 inhibitor (SB-3CT); compared with the Control group, *P < 0.05, **P < 0.01; compared with the OGD / R group, ##P < 0.01; compared with the FMNT group, &&P < 0.01.

[0037] Figure 11FMNT promotes the migration of NSCs after OGD / R by upregulating MMP-2 / 9. Among them, A shows the effects of different treatments on the migration of NSCs from the edge of neurospheres outward; B is the statistical chart of the migration distance of NSCs; scale bar = 100 μm. Compared with the Control group, **P < 0.01; compared with the OGD / R group, ##P < 0.01; compared with the FMNT group, &&P < 0.01.

[0038] Figure 12 AS-IV promotes the expression of PPARγ in NSCs after OGD / R. Among them, A shows the effects of different concentrations of AS-IV on the expression of PPARγ; B shows the effects of AS-IV on the expression of PPARγ after treatment with the PPARγ inhibitor (T0070907); compared with the Control group, **P < 0.01; compared with the OGD / R group, ##P < 0.01; compared with the AS-IV group, &P < 0.05.

[0039] Figure 13 AS-IV promotes the differentiation of NSCs into neurons after OGD / R by upregulating PPARγ. Among them, A shows the immunofluorescence results of different treatment groups; B shows the results of the proportion of NSCs differentiated into neurons (NeuN + ) cells; C shows the results of the proportion of astrocytes (GFAP + ) cells; compared with Control, **P < 0.01; compared with the OGD / R group, ##P < 0.01; compared with the AS-IV group, &&P < 0.01. Detailed implementation methods

[0040] The present invention will be further described below in conjunction with embodiments.

[0041] Example 1

[0042] 1 Experimental materials

[0043] 1.1 Experimental animals

[0044] SD rats (male, SPF grade, 280 - 320 g) were provided by Shanghai Slack Experimental Animal Co., Ltd. [License number: SCXK (Shanghai) 2022 - 0004] and were housed in the Animal Experimental Research Center of Zhejiang Chinese Medical University [License number: SYXK (Zhejiang) 2021 - 0012]. They had free access to water and food.

[0045] 1.2 Main reagents and drugs

[0046] Table 1 Experimental reagents and drugs

[0047]

[0048]

[0049] 2 Methods

[0050] 2.1 Preparation of the middle cerebral artery occlusion / reperfusion (MCAO / R) model in rats

[0051] After anesthetizing the rats, a midline incision about 2 cm long was made in the neck. The common carotid artery, external carotid artery, and internal carotid artery on the right side were bluntly dissected. The distal end of the external carotid artery was ligated and transected with a 6 / 0 suture. The distal ends of the common carotid artery and internal carotid artery were clamped with a microarterial clip. A small incision was made 2 mm from the bifurcation of the external carotid artery, and the MCAO suture was gently inserted through the incision of the external carotid artery and into the origin of the middle cerebral artery through the internal carotid artery. When there was slight resistance at about 18 - 20 mm from the bifurcation of the common carotid artery, the external carotid artery was tied with a 6 / 0 suture to fix the suture. After 90 minutes, the suture was carefully withdrawn, the external carotid artery was ligated, and the skin was sutured after disinfection. During the operation, the rectal temperature of the rats was maintained at about 37 °C with a warming blanket, and the rats were placed in an incubator until they woke up after the operation. The blood vessels of the sham-operated group (Sham) rats were dissected in the same way, but the suture was only inserted about 5 mm.

[0052] 2.2 Animal grouping and administration

[0053] One day after MCAO / R, a modified neurological severity score (mNSS) was performed. Model rats with scores of 10 - 14 were randomly divided into the MCAO / R group, the BYHWD group, and the uniform design group, with 12 - 18 rats in each group. Starting from one day after MCAO / R, the rats in the BYHWD group and the uniform design group (CLG + FMNT + AS-IV-1 - 9, CFA-1 - 9) were given intragastric administration once a day for 14 consecutive days. The rats in the Sham and MCAO / R groups were given intragastric administration of an equal amount of 10% DMSO. The rats in the edaravone group were intraperitoneally injected with edaravone (10 mg / kg) during reperfusion once a day for 14 consecutive days. One day after the operation, BrdU (5-bromodeoxyuridine) (50 mg / kg) was intraperitoneally injected once a day for 14 consecutive days.

[0054] According to the principle of uniform design, the number of levels should be more than twice the number of factors. When selecting the uniform design table, U 9 (9 6 ) uniform design table (Table 2) was selected. At the same time, the number of levels and the arrangement of columns were determined according to the number of factors (Table 3). According to the content of each chemical component in BYHWD determined by the reference literature and previous experiments, the levels (dose ranges) of each component were determined. The specific grouping and doses are shown in Table 4.

[0055] Table 2 U 9 (9 6 ) uniform design table

[0056]

[0057] Table 3 U 9 (9 6 ) Usage table

[0058]

[0059] Table 4 Dosage distribution of each monomer in the uniform design experiment according to the uniform design table U 9 (9 6 )

[0060]

[0061] Note: Each column represents a drug. CFA-1 to CFA-9 are the uniform design groups. Each row represents a formula, with a total of 9 groups; BYHWD: Buyang Huanwu Decoction. ig: intragastric administration; ip: intraperitoneal injection.

[0062] 2.3 Neurological function evaluation

[0063] 2.3.1 Corner test

[0064] The Corner test was used to detect sensorimotor deficits in rats. The corner test device was formed by clamping two identical-sized wooden boards, with the edges of the boards connected at a 30° angle, leaving a gap at the connection. The rats entered through the opening and, after advancing to the narrow corner, their whiskers on both sides would touch the wooden boards, and then they would stand and turn towards the opening end. Normal rats would turn randomly to the left or right, while MCAO / R rats would preferentially turn towards the infarcted side. The number of right turns in 10 trials was recorded.

[0065] 2.3.2 Neurological symptom scoring

[0066] The mNSS was used to evaluate the neurological symptoms of the animals, as shown in Table 5.

[0067] Table 5 mNSS scoring table

[0068]

[0069] 2.5 Statistical analysis

[0070] Data are expressed as mean ± standard deviation Indications. If the data conforms to a normal distribution and homogeneity of variance, the independent samples t-test is used to analyze the differences between two groups, and one-way analysis of variance (One-way ANOVA) is used for multiple groups. The post hoc test uses the Tukey method, and the Tamhane's T2 method is used when the variance is inhomogeneous; for non-normally distributed data, non-parametric tests are used for analysis. The mNSS and corner experiments use non-parametric tests for analysis (Kruskal-Wallis test). A P value < 0.05 is considered statistically significant.

[0071] 3 Experimental results

[0072] As Figure 1 shown, on the 1st day after surgery, compared with the Sham group, the mNSS score and the number of right turns of rats in the MCAO / R group increased significantly (P < 0.05), indicating that the model was successfully established. The mNSS score of rats in the positive drug edaravone group was significantly lower than that in the MCAO / R group. On the 14th day after surgery, compared with the MCAO / R group, the mNSS score and the number of right turns in the CFA-2, CFA-3, CFA-4, CFA-6, CFA-7, and CFA-8 groups decreased significantly, especially in the CFA-6 group. On this basis, using the mNSS scores and the number of right turns of each group in the uniform design on the 14th day after surgery (CFA-1 to CFA-9) weighted (50% each) as the dependent variable (Y), multiple stepwise regression analysis was performed, and the relationship between Y and three components, namely CLG (X 1 ), FMNT (X 2 ), and AS-IV (X 3 ) was obtained. The regression equation was: Y = 8.0664 - 0.0715X 1 - 0.0504X 2 - 0.0346X 3 + 0.0011X 1 * X 1 + 0.0010X 2 * X 2 + 0.0005X 3 * X 3 . According to the equation, the optimal ratio was obtained as X 1 = 32.8 mg / kg / d, X 2 = 25.0 mg / kg / d, X 3 = 34.1 mg / kg / d.

[0073] Example 2 (Pharmacodynamic verification experiment)

[0074] 1 Experimental materials

[0075] The materials used were as shown in Table 1 of Example 1.

[0076] 2 Experimental methods

[0077] 2.1 Grouping and treatment of experimental animals

[0078] One day after MCAO / R surgery, mNSS scores were measured. Model rats with scores of 10 - 14 were randomly divided into the MCAO / R group, the BYHWD group, the best - efficacy group CFA - 6 and the worst - efficacy group CFA - 9 in the uniform - design group in Example 1, the compatibility - optimization group (CFA - optimization, CFA - OP) (i.e., CLG: 32.8 mg / kg / d + FMNT: 25.0 mg / kg / d + AS - IV: 34.1 mg / kg / d in Example 1), and the CFA - OP + T0070907 group (T0070907: 2 mg / kg, ip). The administration route and time were the same as those in the uniform - design experiment in Example 1.

[0079] 2.2 Collection of frozen - section samples

[0080] On the 14th day after MCAO / R surgery in rats, the rats were anesthetized, the chest was opened, and after quickly rinsing with about 250 mL of normal saline through the aorta, they were perfused and fixed with about 250 mL of paraformaldehyde solution (4% PFA). The brain tissues were taken out and left overnight in 4% PFA, and then dehydrated successively with 20% and 30% sucrose solutions. Subsequently, 10 - μm and 20 - μm thick sections were prepared with a cryostat, air - dried, and stored at - 20°C.

[0081] 2.3 Detection of infarction area by toluidine blue staining

[0082] The rat brain sections (20 μm) were rinsed with PBS for 5 min, fixed with 4% PFA for 30 min, stained with 1% toluidine blue solution after PBS rinsing at 37°C for 10 min, rinsed with PBS for 5 min; immersed in 75% alcohol for decolorization for about 20 s, observed the decolorization situation to control the reaction time. Rinsed with PBS for 5 min, dried at 37°C, sealed with neutral balsam, photographed, and the infarction area of the rat brain was corrected and calculated: Corrected infarction area = left - hemisphere area - (right - hemisphere area - right - hemisphere infarction area), Infarction area percentage = corrected infarction area / left - hemisphere area.

[0083] 2.4 Immunofluorescence staining

[0084] Take out rat brain tissue sections (10 μm), rewarm, and fix with 4% PFA for 20 min; incubate with 2 M HCl at 37°C for 30 min. Drop 0.1 M boric acid and incubate at room temperature for 10 min; incubate with 1% Triton X-100 at room temperature for 20 min to permeabilize the membrane; block with 5% donkey serum at 37°C for 1 h. Add primary antibodies BrdU mouse monoclonal antibody (1:200), BrdU sheep polyclonal antibody (1:200), DCX rabbit polyclonal antibody (1:200), NeuN rabbit polyclonal antibody (1:500), GFAP mouse monoclonal antibody (1:200) respectively, and incubate overnight at 4°C. Recover the primary antibody, 5 min each time; drop the corresponding Cy3 or FITC-conjugated fluorescent secondary antibody (1:100), incubate in the dark for 1 h, stain the nucleus with DAPI, and mount the slides. Observe and take pictures under an inverted fluorescence microscope. Randomly select 3 fields of view for each section and count the number of double-positive cells.

[0085] 2.5 Detection of protein expression by Western Blot

[0086] Cell or tissue samples are lysed with RIPA lysis buffer, centrifuged at 13,000 rpm / min at 4°C for 10 min to obtain the supernatant, and the protein concentration is determined by the BCA method. Protein samples are electrophoresed in SDS-PAGE gels, transferred to membranes by wet transfer method, blocked with 5% non-fat milk powder or 5% BSA, incubated with primary antibodies MMP-2 (1:500), MMP-9 (1:1000), p-EGFR (1:1000), EGFR (1:1000), PPARγ (1:500), β-actin (1:1000) overnight, incubated with HRP-labeled goat anti-rabbit or anti-mouse IgG at room temperature for 1 h, developed with ECL luminescent solution, and the gray value of the bands is quantified using ImageJ software.

[0087] 3 Experimental results

[0088] 3.1 Comparison of neurological function recovery in rats of each group after cerebral ischemia

[0089] The results on the 14th day after surgery showed that compared with the Sham group, the mNSS score and the number of right turns in the MCAO / R group were significantly increased. Compared with the MCAO / R group, the mNSS score and the number of right turns in the BYHWD group, CFA-6 and CFA-OP groups of rats were significantly decreased, especially the BYHWD group was the most obvious, and the CFA-OP group was the second ( Figure 2 ). After administration of the PPARγ inhibitor T0070907 (CFA-OP + T007), the mNSS score was significantly increased compared with the CFA-OP group (P < 0.05), and the number of right turns had an upward trend compared with the CFA-OP group, but there was no statistical difference (P > 0.05), see Figure 2 .

[0090] 3.2 Comparison of infarct areas in rats of each group

[0091] On the 14th day after surgery, the cerebral infarction areas of the rats in the BYHWD, CFA-6, and CFA-OP groups were significantly smaller than those in the MCAO / R group. Moreover, the infarction area in the CFA-OP group tended to further decrease compared with that in the CFA-6 group, but the difference between the two was not statistically significant. The infarction area in the CFA-OP + T007 group was significantly increased compared with that in the CFA-OP group( Figure 3 ).

[0092] 3.3 Comparison of neurogenesis in rats of each group after cerebral ischemia

[0093] At 14 days after MCAO / R, newborn NSCs, neural progenitor cells, mature neurons, and astrocytes were detected by double immunofluorescence of BrdU / Nestin, BrdU / DCX, BrdU / NeuN, and BrdU / GFAP, respectively. The immunofluorescence results showed that the number of BrdU + / Nestin + , BrdU + / Nestin + , BrdU + / DCX + , BrdU + / NeuN + , BrdU + / GFA + positive cells in the MCAO / R group was significantly increased compared with that in the Sham group; the BYHWD and CFA-6 groups were significantly increased compared with the MCAO / R group. The CFA-OP group was further increased compared with the CFA-6 group( Figure 4 ), and there was no significant difference from the BYHWD group. Compared with the CFA-OP group, the number of BrdU + / Nestin + , BrdU + / DCX + , BrdU + / NeuN + , BrdU + / GFAP + positive cells in the CFA-OP + T007 group was significantly decreased compared with that in the CFA-OP group( Figures 4 to 7 ).

[0094] Study on the mechanism of the effective ingredient compound in Example 3 promoting neurogenesis after cerebral ischemia

[0095] 1 Experimental materials

[0096] The test drugs and reagents used are shown in Table 6, and the rest are the same as in Example 1.

[0097] Table 6 Experimental drugs and reagents

[0098]

[0099]

[0100] 2 Experimental methods

[0101] 2.1 Cell culture and establishment of oxygen-glucose deprivation / reperfusion model

[0102] Implemented according to the method in the reference (Li Yu, Ding Wenqian, Li Lin, et al. Astragaloside IV up-regulates miR-199a-5p expression to inhibit oxygen-glucose deprivation / reperfusion-induced apoptosis of neural stem cells [J]. Journal of Zhejiang Chinese Medical University, 2022, 46(05): 473-482.). Specifically, neonatal mice were euthanized and disinfected with 75% ethanol. The heads of neonatal mice were cut off, and the brain tissues were taken out. The meninges and blood vessels were quickly dissected, the cerebral cortex was separated, and 0.25% trypsin digestive solution was added and digested at 37 °C for 15 minutes, then the digestion was terminated. Gently pipette until no tissue fragments were seen, and pass through a 200-mesh filter. Centrifuge at 1000 rpm for 10 min, resuspend, centrifuge again, add an appropriate amount of NSCs complete medium and pipette to resuspend, inoculate into a T25 culture flask, and place in an incubator at 37 °C and 5% CO 2 for culture. Change the medium after 2 days, and then change the culture medium every 1-2 days. Primary NSCs were cultured for about 1 week and then passaged. Collect the cells, centrifuge at 800 rpm for 5 min, discard the supernatant, add Accutase enzyme (0.5 mL per flask) and gently pipette several times, digest at 37 °C for 5 min. Centrifuge at 800 rpm for 5 min at room temperature to completely remove Accutase, add 1 mL of medium to resuspend, continuously pipette with a pipette tip to make a single-cell suspension, passage at a ratio of 1:2, and take the cells of the 3rd to 5th generations for experiments.

[0103] After digesting NSCs into a single-cell suspension with Accutase enzyme, inoculate them into a 96-well plate coated with poly-D-lysine (PDL) (2×104 / well), culture for 24 h, then change to DMEM sugar-free medium and place in an anaerobic chamber, and ventilate with a ternary mixture of 94% N 2 、5% CO 2 、1% O 2 for 6 min, and anaerobically culture at 37 °C for 6 h. Then change to NSCs complete medium and restore to normal culture conditions for reoxygenation for 24 h. The normal control group (Control) was cultured under normal conditions for the same time.

[0104] 2.2 Grouping of cell experiments and intervention methods

[0105] 2.2.1 CLG promotes the proliferation of NSCs after OGD / R

[0106] The primary NSCs were digested into single-cell suspensions and seeded in 96-well or 6-well plates coated with PDL (2×10 5 / mL) and cultured for 24 h. The NSCs were divided into a Control group, an OGD / R group, a CLG group (12.5 μM), and a CLG + AG1478 group. The CLG + AG1478 group was pretreated with the EGFR inhibitor AG1478 (10 μM) for 1 h. After OGD / R treatment, at the time of reoxygenation, the corresponding CLG was added to the CLG group and the CLG + AG1478 group for continued culture for 1 h. The Control group was treated with the corresponding solvent for the same time. Cells were collected and total protein was extracted. At the time of reoxygenation, BrdU (10 μM) was added simultaneously and cultured for 24 h for detecting cell proliferation by immunofluorescence assay.

[0107] 2.2.2 FMNT promotes the migration of NSCs after OGD / R

[0108] The NSCs were digested into neurospheres with a diameter of approximately 200 μm and seeded in 48-well plates coated with PDL for overnight culture to allow the neurospheres to attach. The NSCs were divided into a Control group, an OGD / R group, an FMNT group (100 μM), and an FMNT + SB-3CT group (10 μM). The FMNT + SB-3CT group was pretreated with the MMP2 / 9 inhibitor SB-3CT (10 μM) for 2 h. At the time of reoxygenation, the FMNT group and the FMNT + SB-3CT group were added with FMNT (100 μM) and cultured for 24 h.

[0109] 2.2.3 AS-IV promotes the differentiation of NSCs after OGD / R

[0110] The primary NSCs were digested into single-cell suspensions and seeded in 96-well or 6-well plates coated with PDL (2×10 5 / mL) and cultured for 24 h. The NSCs were divided into a Control group, an OGD / R group, an AS-IV group (25 μM), and an AS-IV + T0070907 group. The AS-IV + T0070907 group was pretreated with T0070907 (15 μM) for 30 min. At the time of reoxygenation, complete medium containing AS-IV or T0070907 was added. After 24 h of reoxygenation, it was changed to differentiation medium containing AS-IV and continued to be cultured for 7 days for subsequent experiments.

[0111] 3 Experimental results

[0112] 3.1 CLG promotes the proliferation of NSCs after OGD / R by upregulating the phosphorylation level of EGFR

[0113] The results suggested that CLG promoted the phosphorylation of EGFR in a concentration-dependent manner, and the effect of 12.5 μM was the best ( Figure 8A). Pretreatment with AG-1478 (10 μM) could significantly reverse the phosphorylation of EGFR induced by CLG. Figure 8 B). Immunofluorescence results showed that 12.5 μM of CLG significantly increased the proportion of BrdU + / Nestin + cells after OGD / R, while pretreatment with AG-1478 significantly reversed the effect of CLG. Figure 9 ).

[0114] 3.2 FMNT promotes the migration of NSCs after OGD / R by upregulating MMP-2 / 9

[0115] The results showed that FMNT promoted the expression of MMP-2 and MMP-9 in a concentration-dependent manner, and the effect of 100 μM was the best. Figure 10 A, B). Pretreatment with the MMP-2 / 9 inhibitor SB-3CT (10 μM) could significantly reverse the expression of MMP-2 / 9 induced by FMNT. Figure 10 C, D), and significantly reverse the effect of FMNT in promoting the migration of NSCs after OGD / R. Figure 11 ).

[0116] 3.3 AS-IV promotes the differentiation of NSCs into neurons after OGD / R by upregulating PPARγ

[0117] AS-IV promoted the expression of PPARγ in a concentration-dependent manner, and the effect of 25 μM was the best. Figure 12 A), and pretreatment with the PPARγ inhibitor T007 (15 μM) could significantly reverse the expression of PPARγ induced by AS-IV. Figure 12 B). Immunofluorescence results showed that the proportion of NSCs differentiated into neurons (NeuN + ) cells in the AS-IV (25 μM) group was significantly higher than that in the OGD / R group, while the proportion of astrocytes (GFAP + ) cells was significantly lower, and pretreatment with T007 could significantly reverse the effect of AS-IV. Figure 13 ).

[0118] In summary, this traditional Chinese medicine multi-component compound significantly alleviated neurological deficits, reduced the area of cerebral infarction, promoted neurogenesis after cerebral infarction, and improved the prognosis of cerebral infarction through multiple pathways and multiple targets.

[0119] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A Chinese medicine active ingredient composition for treating cerebral infarction, characterized in that: The effective ingredients of the traditional Chinese medicine active ingredient composition include calycosin, formononetin and astragaloside IV.

2. The Chinese medicinal active ingredient composition for treating cerebral infarction according to claim 1, characterized in that: The dosage of the Chinese medicine active ingredient composition is 1-33 mg / kg per day for calycosin, 0.2-25.6 mg / kg per day for formononetin, and 1.25-100 mg / kg per day for astragaloside IV.

3. The Chinese medicinal active ingredient composition for treating cerebral infarction according to claim 2, characterized in that: The dosage of the Chinese medicine active ingredient composition is 21-33 mg / kg per day for calycosin, 6.4-25.6 mg / kg per day formononetin, and 20-60 mg / kg per day for astragaloside IV.

4. The Chinese medicinal active ingredient composition for treating cerebral infarction according to claim 1, characterized in that: The dosage form of the medicine is liquid, powder injection, tablet, capsule, powder, pill, oral liquid, paste, granule or dressing.

5. Use of the Chinese medicinal active ingredient composition according to any one of claims 1 to 4 in the preparation of a medicament for treating cerebral infarction.

6. The use according to claim 5, characterized in that: The dosage form of the medicine is liquid, powder injection, tablet, capsule, powder, pill, oral liquid, paste, granule or dressing.

7. The use according to claim 6, characterized in that: When the dosage form of the drug is a liquid, the administration method includes oral administration; The dosage of the calycosin in the medicine is 1-33 mg / kg per day, the dosage of formononetin is 0.2-25.6 mg / kg per day, and the dosage of astragaloside IV is 1.25-100 mg / kg per day.

8. The use according to claim 7, characterized in that: The dosage of the calycosin in the medicine is 21-33 mg / kg per day, the dosage of formononetin is 6.4-25.6 mg / kg per day, and the dosage of astragaloside IV is 20-60 mg / kg per day.

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