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

Abstract

The application discloses a traditional Chinese medicine active ingredient composition for treating cerebral infarction and application thereof, and relates to the technical field of traditional Chinese medicine.The application optimizes the dosage ratio of active ingredient compounds (calycosin-7-glucoside, formononetin and astragaloside IV), proves that the compound can significantly reduce nerve function defect, reduce the cerebral infarction area, promote neurogenesis after cerebral infarction, and effectively improve the prognosis of cerebral infarction, and the effect is equivalent to that of a clinical prescription, i.e., Buyang Huanwu Decoction.The active ingredient composition obtained by the application has the advantages of clear components, clear mechanism and controllable quality, improves the safety of clinical medication on the basis of ensuring the curative effect, reduces the hidden danger of adverse reactions, and is beneficial to the preparation of modern compound traditional Chinese medicine with active ingredient compounding in a modern dosage form.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a composition of active ingredients of traditional Chinese medicine for treating cerebral infarction and its application. Background Technology

[0002] Currently, stroke is the second leading cause of death and the third leading cause of disability worldwide, severely impacting patients' quality of life and increasing the economic burden of healthcare. Clinically, stroke can be divided into ischemic stroke and hemorrhagic stroke, with approximately 80% being ischemic stroke, also known as cerebral infarction. The pathophysiological mechanism of ischemic stroke is complex. In the acute phase, cerebral hypoxia and ischemia mainly lead to intracellular calcium overload, neurotransmitter release disorders, apoptosis, and neuronal necrosis; while the subacute and chronic phases are mainly characterized by neuronal repair and functional reconstruction. Intravenous thrombolysis and endovascular therapy are the two primary strategies for the treatment of acute cerebral infarction in clinical practice. However, these have disadvantages such as a narrow treatment window, hemorrhagic transformation, and reperfusion injury, limiting the number of patients who benefit clinically. Furthermore, due to the poor hypoxia tolerance of nerve cells, even if blood vessels are reopened, neuronal death in the infarct core area is inevitable, leading to sequelae in patients. Because the pathophysiological mechanism of ischemic brain injury is very complex, most clinical trials of neuroprotective drugs developed for ischemic brain injury to date have ended in failure. However, nerve repair not only promotes the reconstruction of neurological function after stroke, but also has a longer therapeutic window. Therefore, discovering drugs that can promote nerve repair after cerebral infarction is of great clinical significance.

[0003] Neural development plays a crucial role in neurological repair during both the subacute and chronic phases following stroke and has attracted increasing attention. Numerous studies have shown that regulating endogenous neurogenesis can improve neurological function repair after stroke, while inhibiting neurogenesis can worsen post-stroke neurological deficits and hinder recovery. Transplantation of neural progenitor cells can improve neurological function. However, the biological processes of neurogenesis are complex, involving multiple stages such as the proliferation, migration, differentiation, and axonal regeneration of neural stem cells (NSCs). Therefore, intervention targeting a single factor cannot completely repair neurological function after stroke.

[0004] Traditional Chinese medicine believes that qi deficiency and blood stasis are the main pathogenesis of cerebral infarction, and the treatment focuses on tonifying qi, activating blood circulation, and unblocking the meridians. Buyang Huanwu Decoction (BYHWD) is a representative formula in traditional Chinese medicine for treating cerebral infarction. It consists of Astragalus membranaceus, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, Carthamus tinctorius, Prunus persica, and Pheretima aspergillum. Its key feature is the combination of a large dose of the qi-tonifying herb Astragalus membranaceus with a small dose of the blood-activating and stasis-removing herbs. This combination promotes qi and blood circulation without harming the body's vital energy, achieving the combined effect of tonifying qi, activating blood circulation, and unblocking the meridians. Previous studies in our laboratory have found that BYHWD promotes the long-term recovery of sensory, motor and learning memory functions in rats with cerebral ischemia (Chu Lisheng, Yang Wuming, Shao Liang, et al. Effects of Buyang Huanwu Decoction on learning and memory function 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 effect of Buyang Huanwu Decoction on focal cerebral ischemia injury in rats. Chinese Journal of Clinical Rehabilitation 2006; 10(11): 56-58.). Further research found that BYHWD promotes angiogenesis, neurogenesis, axonal regeneration, and synaptic plasticity after cerebral ischemia (Chu Lisheng, Jiang Yanyan, Ke Qing, et al. Buyang Huanwu Decoction promotes angiogenesis and functional recovery in rats after focal cerebral ischemia. Chinese Journal 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 mice with focal cerebral ischemia. 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 angiogenesis and Ang-1 / Tie-2 expression in rats). 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. Journal of Zhejiang University of Traditional Chinese Medicine, 2011, 35(3): 375-377. Liu Zhiting, Yu Tianhong, Qu Tiebing, et al. Effects of Buyang Huanwu Decoction on the expression of growth-related protein 43 and synaptophysin 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 region. Chinese Journal of Traditional Chinese Medicine, 2015; 40(2): 298-302. Through decomposition studies, it was found that the promotion of neurogenesis after cerebral infarction by BYHWD is mainly closely related to the high dose of Qi-tonifying 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 decompositions 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 efficacy through multiple components, multiple targets, and multiple pathways through combination, which has obvious advantages over drugs with single components and single targets.However, the traditional Chinese medicine decoction pieces have numerous chemical components and their mechanisms of action are difficult to define. At the same time, the origin of the medicinal materials, storage and maintenance conditions, and processing methods may lead to different therapeutic effects, which limits their clinical application, promotion, and international and modern development.

[0005] To better leverage the advantages of traditional Chinese medicine (TCM), in-depth research is needed on the pharmacodynamic substances and mechanisms of action in TCM prescriptions. The compatibility analysis of effective components in TCM, based on compatibility theory and combined with modern technology, involves identifying effective ingredients, confirming their efficacy, optimizing dosage, and studying mechanisms to achieve the goals of reducing toxicity and enhancing efficacy. Originating from TCM, this approach ensures definite pharmacodynamic substances and controllable quality. Furthermore, it is suitable for studying its mechanisms of action at the animal and cellular levels, possessing both the advanced features of modern pharmaceutical manufacturing and retaining the traditional characteristics of TCM compatibility analysis, thus providing new insights for the innovative development of TCM. Summary of the Invention

[0006] In view of this, the present invention discloses a composition of active ingredients of traditional Chinese medicine for treating cerebral infarction and its application. The composition of active ingredients of traditional Chinese medicine in the present invention (verrucoside, styracoside and astragaloside A) and the optimal combination of active ingredients in different proportions are obtained. It has the characteristics of promoting nerve repair after cerebral infarction through multiple components, multiple pathways and multiple effects.

[0007] A series of pathophysiological responses triggered by cerebral infarction, including inflammation, oxidative stress, and blood-brain barrier disruption, exacerbate brain tissue damage but also initiate a degree of self-repair mechanisms. Endogenous neurogenesis is a crucial step in post-infarction neural repair. Endogenous neurogenesis refers to the proliferation, migration, and differentiation of neural stem cells (NSCs) around the infarct lesion. These newly generated neurons and supporting cells can partially replace damaged cells and participate in the reconstruction of neural networks. However, these endogenous repair capabilities are usually insufficient to fully restore damaged neural function. In summary, post-infarction neural repair is a complex, multifactorial process involving neurogenesis, angiogenesis, and inflammatory regulation. In-depth research into its mechanisms and the development of effective interventions are of great significance for improving the prognosis of patients with cerebral infarction.

[0008] This invention provides a traditional Chinese medicine active ingredient composition for treating cerebral infarction, wherein the effective components of the traditional Chinese medicine active ingredient composition include Calycosin-7-glucoside (CLG), Formononetin (FMNT), and Astragaloside IV (AS-IV).

[0009] Verbena isoflavone glycosides are flavonoid compounds found primarily in legumes. They are one of the quality control indicators for Astragalus membranaceus and possess antioxidant and anti-apoptotic activities, exhibiting some protective effects on nerve cells. The molecular formula of verbena isoflavone glycosides is C2. 22 H 22 O 10 CAS No.: 20633-67-4, Chemical Formula as follows:

[0010]

[0011] Mangosteen flavonoids are natural flavonoids widely found in various plants, especially legumes such as Astragalus membranaceus. They possess multiple pharmacological effects, including anti-inflammatory, antioxidant, and neuroprotective properties. The molecular formula of mangosteen flavonoids is C2. 16 H 12 O4, CAS No.: 485-72-3, chemical formula as follows:

[0012]

[0013] Astragaloside A is a natural saponin compound and the main active ingredient in Astragalus membranaceus. Due to its multiple pharmacological effects, it exhibits significant therapeutic potential in areas such as immunomodulation, anti-inflammation, anti-oxidation, cardiovascular protection, and neuroprotection. The molecular formula of astragaloside A is C2. 41 H 68 O 14 CAS No.: 84687-43-4, Chemical Formula as follows:

[0014]

[0015] Specifically, the dosage of verbascoside in the active ingredient composition of the traditional Chinese medicine is 1-33 mg / kg per day, the dosage of styracin is 0.2-25.6 mg / kg per day, and the dosage of astragaloside A is 1.25-100 mg / kg per day.

[0016] More preferably, the dosage of verbascoside, mangiferin, and astragaloside A in the active ingredient composition of the traditional Chinese medicine is 21-33 mg / kg per day, 6.4-25.6 mg / kg per day, and 20-60 mg / kg per day.

[0017] Most preferably, the dosage of verbascoside, mangiferin, and astragaloside A in the active ingredient composition of the traditional Chinese medicine is 32.8 mg / kg per day, 25.0 mg / kg per day, and 34.1 mg / kg per day.

[0018] The present invention also provides the application of the aforementioned 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, powder for injection, tablet, capsule, powder, pill, oral liquid, ointment, granule or dressing.

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

[0021] The dosage of the drug is 1-33 mg / kg per day for verbascoside, 0.2-25.6 mg / kg per day for astragaloside A, and 1.25-100 mg / kg per day for astragaloside A.

[0022] More preferably, the dosage of verrucoside isoflavone glycoside in the pharmaceutical composition is 21-33 mg / kg per day, the dosage of styracin is 6.4-25.6 mg / kg per day, and the dosage of astragaloside A is 20-60 mg / kg per day.

[0023] Most preferably, the dosage of verbascoside in the drug is 32.8 mg / kg per day, the dosage of styracin is 25.0 mg / kg per day, and the dosage of astragaloside A is 34.1 mg / kg per day.

[0024] This invention relates to a compound pharmaceutical composition consisting of three active ingredients from traditional Chinese medicine: verrucoside, gentianin, and astragaloside A. The inventors, utilizing key technologies for quantitative design of traditional Chinese medicine compound formulas, predicted the optimal dosage ratio for efficacy through uniform design experiments and mathematical models. Animal experiments were then conducted to verify the efficacy, yielding the optimal combination. This method is reasonable and feasible. The composition of this invention has clearly defined components and controllable quality. While ensuring efficacy, it also reduces the dosage, improving the safety of clinical use and minimizing the risk of adverse reactions. This facilitates the development of modern compound traditional Chinese medicines with active ingredient combinations in modern dosage forms, exhibiting multi-component, multi-pathway, and multi-effect therapeutic effects for cerebral infarction.

[0025] The beneficial effects of this invention are:

[0026] This invention, through optimization of the dosage ratio of the active ingredient compound, demonstrates that this compound (especially CLG 32.8 mg / kg, FMNT 25.0 mg / kg, and AS-IV 34.1 mg / kg, once daily) significantly reduces neurological deficits, decreases the infarct area, and promotes post-infarction neurogenesis, effectively improving the prognosis of cerebral infarction. The effects are comparable to those of the clinically proven formula "Buyang Huanwu Decoction." The active ingredient composition obtained by this invention has the advantages of clear components, well-defined mechanisms, and controllable quality. While ensuring efficacy, it also improves the safety of clinical use, reduces the risk of adverse reactions, and facilitates the development of modern compound traditional Chinese medicines with active ingredient formulations in modern dosage forms. Attached Figure Description

[0027] Figure 1 To investigate the effects of each treatment on the recovery of neurological function after cerebral ischemia in rats using a uniform design, A is a line graph of the rat mNSS score; B is the rat horn test result; Sham: sham-operated group; MCAO / R: model group; Edaravone: edaravone group; CFA-1~9: uniform design groups 1~9.

[0028] Figure 2 To uniformly design and verify the effect of each experimental group on the recovery of neurological function after cerebral ischemia in rats; where A is the mNSS score and B is the number of right turns.

[0029] Figure 3 The effect of each treatment group on infarct area is shown in Figure A. A represents a stained section of rat brain tissue, and B represents a statistical graph of 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 enhance the effective active ingredients of BYHWD, increase BrdU in rats with cerebral ischemia. + / Nestin + Cell count; where A is an immunofluorescence staining image of a rat brain tissue section; B is a BrdU staining image of a rat with cerebral ischemia. + / Nestin + Cell count; 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 enhance the effective active ingredients of BYHWD, increase BrdU in rats with cerebral ischemia. + / DCX + Cell count; where A is an immunofluorescence staining image of a rat brain tissue section; B is a BrdU staining image of a rat with cerebral ischemia. + / DCX + Cell count; *P<0.05 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.

[0032] Figure 6 To enhance the effective active ingredients of BYHWD, increase BrdU in rats with cerebral ischemia. + / NeuN + Cell count; where A is an immunofluorescence staining image of a rat brain tissue section; B is a BrdU staining image of a rat with cerebral ischemia. + / NeuN+ Cell count; 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 To enhance the effective active ingredients of BYHWD, increase BrdU in rats with cerebral ischemia. + / GFAP + Cell count; where A is an immunofluorescence staining image of a rat brain tissue section; B is a BrdU staining image of a rat with cerebral ischemia. + / GFAP + Cell count; 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 p-EGFR expression in NSCs after OGD / R; where A represents the effect of different concentrations of CLG on EGFR phosphorylation; B represents 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 NSC proliferation after OGD / R by upregulating EGFR phosphorylation levels; where A represents the immunofluorescence assay results of CLG-induced NSC proliferation after OGD / R; B represents BrdU. + / Nestin + Changes in cell proportions; compared with Control group, **P<0.01; compared with OGD / R group, ##P<0.01; compared with CLG group, &&P<0.01.

[0036] Figure 10 To promote the expression of MMP-2 / 9 in NSCs after OGD / R; where A and B represent the effects of different concentrations of FMNT on the expression of MMP-2 and MMP-9; C and D represent 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 up-regulating MMP-2 / 9; wherein, A shows the effect of different treatments on the outward migration of NSCs from the edge of neurospheres; B is a statistical diagram 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; wherein, A shows the effect of different concentrations of AS-IV on PPARγ expression; B shows the effect of AS-IV on PPARγ expression after treatment with 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 neuronal differentiation of NSCs after OGD / R by up-regulating PPARγ; wherein, A is the immunofluorescence results of different treatment groups; B shows that NSCs differentiate into neurons (NeuN + ) cell proportion results; C is astrocytes (GFAP + ) cell proportion results; compared with Control, **P < 0.01; compared with OGD / R group, ##P < 0.01; compared with AS-IV group, &&P < 0.01. Detailed Description of Embodiments

[0040] The present invention is further described below with reference to examples.

[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 Laboratory Animal Co., Ltd. [License No.: SCXK (Hu) 2022-0004], and housed in the Animal Experimental Research Center of Zhejiang Chinese Medical University [License No.: SYXK (Zhe) 2021-0012]. 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 Establishment of a rat model of middle cerebral artery occlusion / reperfusion (MCAO / R)

[0051] After anesthetizing the rats, a midline incision of approximately 2 cm was made in the neck. The right common carotid, external carotid, and internal carotid arteries were bluntly dissected. The distal end of the external carotid artery was ligated and severed with 6 / 0 sutures. Micro-arterial clips were used to clamp the distal end of the common carotid artery and the internal carotid artery. A small incision was made 2 mm from the bifurcation of the external carotid artery. The MCAO suture was gently inserted through the incision in the external carotid artery, passing through the internal carotid artery to the origin of the middle cerebral artery. When slight resistance was encountered approximately 18-20 mm from the bifurcation of the common carotid artery, the suture was secured to the external carotid artery with 6 / 0 sutures. After 90 minutes, the suture was carefully removed, the external carotid artery was ligated, and the skin was sutured after disinfection. During the operation, the rats' rectal temperature was maintained at approximately 37°C using a warming blanket. Postoperatively, the rats were placed in a constant-temperature incubator until they regained consciousness. The blood vessels of the sham-operated group (Sham) rats were dissected using the same method, but the suture was only inserted to approximately 5 mm.

[0052] 2.2 Animal grouping and administration

[0053] One day after MCAO / R surgery, a modified neurological severity score (mNSS) was performed. Model rats with scores of 10-14 were randomly divided into MCAO / R group, BYHWD group, and uniform design group, with 12-18 rats in each group. Starting one day after MCAO / R surgery, the BYHWD group and the uniform design group (CLG+FMNT+AS-IV-1~9, CFA-1~9) were administered the drug via gavage once daily for 14 days. Rats in the Sham and MCAO / R groups were administered an equal volume of 10% DMSO via gavage. Rats in the edaravone group received intraperitoneal injection of edaravone (10 mg / kg) once daily for 14 days upon reperfusion. One day after surgery, BrdU (5-bromodeoxyuridine) (50 mg / kg) was administered intraperitoneally once daily for 14 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, choose U9(9). 6 A uniform design table (Table 2) was used, and the number of levels and column arrangement were determined based on the number of factors (Table 3). The levels (dosage ranges) of each component were determined based on the content of each chemical component in BYHWD as determined by references and previous experiments. The specific groupings and dosages are shown in Table 4.

[0055] Table 2 U9(9) 6 Uniform Design Table

[0056]

[0057] Table 3 U9(9) 6 Usage table

[0058]

[0059] Table 4. Uniform Design Experiments: Each monomer is designed according to table U9(9). 6 ) Distributed dose

[0060]

[0061] Note: Each column represents one drug. CFA-1 to CFA-9 are uniform design groups. Each row represents one formulation, for a total of 9 groups. BYHWD: Buyang Huanwu Decoction. ig: gavage; ip: intraperitoneal injection.

[0062] 2.3 Neurological Function Evaluation

[0063] 2.3.1 Angle Experiment

[0064] The Corner test was used to assess sensorimotor impairment in rats. The Corner test apparatus consisted of two identical wooden boards joined at a 30° angle, with a gap at the joint. The rat entered through the opening, advanced into the narrow corner until its whiskers touched the boards, then stood up and turned to face the opening. Normal rats randomly turned left or right, while MCAO / R rats preferentially turned to the infarct side. The number of right turns was recorded in 10 trials.

[0065] 2.3.2 Neurological Symptom Scores

[0066] The neurological symptoms of the animals were assessed using mNSS, as shown in Table 5.

[0067] Table 5mNSS Scoring Table

[0068]

[0069] 2.5 Statistical Analysis

[0070] Data expressed as mean ± standard deviation The data are presented as follows. If the data conforms to a normal distribution and has homogeneity of variance, the difference between two groups is analyzed using an independent samples t-test; differences among multiple groups are analyzed using one-way ANOVA, with the Tukey method used for post-tests; and Tamhane's T² method is used when variances are unequal. For non-normally distributed data, nonparametric tests are used for analysis. mNSS and angle experiments are analyzed using nonparametric tests (Kruskal-Wallis test). A p-value < 0.05 is considered statistically significant.

[0071] 3 Experimental Results

[0072] like Figure 1 As shown, on postoperative day 1, compared with the Sham group, the mNSS score and number of right turns in the MCAO / R group rats were significantly increased (P < 0.05), indicating a successful model. The mNSS score of rats in the positive drug edaravone group was significantly lower than that in the MCAO / R group. On postoperative day 14, compared with the MCAO / R group, the mNSS score and number of right turns in the CFA-2, CFA-3, CFA-4, CFA-6, CFA-7, and CFA-8 groups were significantly reduced, with the CFA-6 group showing the most significant effect. Based on this, a multivariate stepwise regression analysis was performed with a uniform design of mNSS score and number of right turns (50% each) in each group (CFA-1 to CFA-9) as the dependent variable (Y) on postoperative day 14, to obtain the relationship between Y and three components, namely CLG (X1), FMNT (X2), and AS-IV (X3). The regression equation is: Y = 8.0664 - 0.0715X1 - 0.0504X2 - 0.0346X3 + 0.0011X1*X1 + 0.0010X2*X2 + 0.0005X3*X3. Based on the equation, the optimal ratio is X1 = 32.8 mg / kg / d, X2 = 25.0 mg / kg / d, and X3 = 34.1 mg / kg / d.

[0073] Example 2 (Pharmacodynamic Validation Experiment)

[0074] 1. Experimental Materials

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

[0076] 2 Experimental Methods

[0077] 2.1 Grouping and treatment of experimental animals

[0078] One day after MCAO / R surgery, the mNSS score was assessed. Model rats with scores of 10-14 were randomly divided into the MCAO / R group, the BYHWD group, and the best-efficacy group CFA-6 and the worst-efficacy group CFA-9 from the uniform design group in Example 1, the compatibility optimization group (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 route and time of administration were the same as in the uniform design experiment in Example 1.

[0079] 2.2 Collection of frozen section samples

[0080] On day 14 after MCAO / R surgery in rats, the rats were anesthetized, the thoracic cavity was opened, and the tissue was rapidly flushed with approximately 250 mL of physiological saline via the aorta, followed by fixation with approximately 250 mL of paraformaldehyde solution (4% PFA). Brain tissue was removed and incubated overnight in 4% PFA, then dehydrated sequentially with 20% and 30% sucrose solutions. Subsequently, sections of 10 μm and 20 μm thickness were prepared using a cryostat, air-dried, and stored at -20°C.

[0081] 2.3 Toluidine blue staining to detect infarct area

[0082] Rat brain sections (20 μm) were rinsed with PBS for 5 min, fixed with 4% PFA for 30 min, rinsed with PBS, stained with 1% toluidine blue solution at 37℃ for 10 min, rinsed with PBS for 5 min, and then destained with 75% alcohol for about 20 s. The fading was observed and the reaction time was controlled. After rinsing with PBS for 5 min, the sections were dried at 37℃, mounted with neutral resin, photographed, and the infarct area of ​​the rat brain was corrected and calculated: Corrected infarct area = Left hemisphere area - (Right hemisphere area - Right hemisphere infarct area), and the percentage of infarct area = Corrected infarct area / Left hemisphere area.

[0083] 2.4 Immunofluorescence staining

[0084] Rat brain tissue sections (10 μm) were removed, rewarmed, and fixed with 4% PFA for 20 min; then incubated with 2M HCl at 37℃ for 30 min. 0.1M boric acid was added, and the sections were incubated at room temperature for 10 min; the membrane was ruptured by incubation with 1% Triton X-100 at room temperature for 20 min; and blocked with 5% donkey serum at 37℃ for 1 h. Primary antibodies against BrdU mouse monoclonal antibody (1:200), BrdU sheep polyclonal antibody (1:200), DCX rabbit polyclonal antibody (1:200), NeuN rabbit polyclonal antibody (1:500), and GFAP mouse monoclonal antibody (1:200) were added, and incubated overnight at 4℃. The primary antibodies were recovered, and each time for 5 min; the corresponding Cy3 or FITC-conjugated fluorescent secondary antibody (1:100) was added, and the sections were incubated in the dark for 1 h, followed by DAPI staining of the nuclei and mounting. The sections were observed and photographed under an inverted fluorescence microscope. Three fields of view were randomly selected from each section, and the number of double-positive cells was counted.

[0085] 2.5 Western Blot detection of protein expression

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

[0087] 3 Experimental Results

[0088] 3.1 Comparison of neurological function recovery after cerebral ischemia in different groups of rats

[0089] Results on postoperative day 14 showed that, compared with the Sham group, the MCAO / R group had significantly higher mNSS scores and more right turns. Compared with the MCAO / R group, the BYHWD, CFA-6, and CFA-OP groups showed significantly lower mNSS scores and more right turns, especially the BYHWD group, followed by the CFA-OP group. Figure 2 ), administration of the PPARγ inhibitor T0070907 (CFA-OP+T007) significantly increased the mNSS score compared to the CFA-OP group (P<0.05), and the number of right turns showed an increasing trend compared to the CFA-OP group, however, there was no statistical difference (P>0.05). (See [link to relevant documentation]). Figure 2 .

[0090] 3.2 Comparison of infarct area among rats in different groups

[0091] On postoperative day 14, the infarct area in the BYHWD, CFA-6, and CFA-OP groups was significantly smaller than that in the MCAO / R group, and the infarct area in the CFA-OP group showed a further decreasing trend compared to the CFA-6 group, but the difference between the two groups was not statistically significant. The infarct area in the CFA-OP+T007 group was significantly larger than that in the CFA-OP group. Figure 3 ).

[0092] 3.3 Comparison of neurogenesis after cerebral ischemia in different groups of rats

[0093] Fourteen days after MCAO / R surgery, BrdU / Nestin, BrdU / DCX, BrdU / NeuN, and BrdU / GFAP immunofluorescence dual staining were used to detect newly formed NSCs, neural progenitor cells, mature neurons, and astrocytes, respectively. Immunofluorescence results showed that BrdU in the MCAO / R group... + / Nestin + ,BrdU+ / Nestin + 、BrdU + / DCX + 、BrdU + / NeuN + 、BrdU + / GFA + The number of P cells was significantly increased in the BYHWD and CFA-6 groups compared to the MCAO / R group. The CFA-OP group showed a further increase compared to the CFA-6 group. Figure 4 The levels of BrdU in the CFA-OP+T007 group were significantly different from those in the BYHWD group. Compared to the CFA-OP group, the BrdU levels in the CFA-OP+T007 group were significantly higher than those in the CFA-OP group. + / Nestin + 、BrdU + / DCX + 、BrdU + / NeuN + 、BrdU + / GFAP + The number of cells decreased significantly. Figures 4-7 ).

[0094] Example 3: Study on the mechanism by which the compound active ingredients promote 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 an oxygen-glucose deprivation / reperfusion model

[0102] The procedure was performed according to the method described in the literature (Li Yu, Ding Wenqian, Li Lin, et al. Astragaloside A upregulation of miR-199a-5p expression inhibits oxygen-glucose deprivation / reperfusion-induced apoptosis of neural stem cells [J]. Journal of Zhejiang University of Traditional Chinese Medicine, 2022, 46(05): 473-482.). Specifically, the suckling mice were euthanized and disinfected with 75% ethanol. The heads of the suckling mice were cut off, and the brain tissue was removed. The meninges and blood vessels were quickly dissected, the cerebral cortex was separated, and 0.25% trypsin digestion solution was added. Digestion was stopped after 15 minutes at 37°C. The tissue was gently blown away with a dropper until no tissue fragments were visible, and then filtered through a 200-mesh sieve. The tissue was centrifuged at 1000 rpm for 10 minutes, resuspended, centrifuged again, and resuspended with an appropriate amount of NSCs complete culture medium. The tissue was then inoculated into T25 culture flasks and placed in a 37°C, 5% CO2 incubator. The culture medium was changed after 2 days, and then changed every 1-2 days thereafter. Primary NSCs were cultured for approximately one week before passage. Cells were collected, centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. Accutase enzyme (0.5 mL per flask) was added, and the cells were gently pipetted several times. The cells were then digested at 37°C for 5 minutes. After centrifugation at 800 rpm for 5 minutes at room temperature to completely remove the Accutase, the cells were resuspended in 1 mL of culture medium and continuously pipetted to prepare a single-cell suspension. Cells were passaged at a ratio of 1:2, and cells from passages 3 to 5 were used for experiments.

[0103] NSCs were digested into single-cell suspensions using Accutase enzyme and then seeded into 96-well plates (2 × 10⁴ cells / well) coated with poly-D-lysine hydrobromide (PDL). After 24 h of culture, the medium was replaced with DMEM sugar-free medium and placed in an anoxic chamber. Aeration was provided for 6 min with a ternary gas mixture of 94% N₂, 5% CO₂, and 1% O₂, and the cells were kept at 37°C for 6 h under anoxic conditions. Then, the medium was replaced with complete NSC culture medium, and the cells were reoxygenated for 24 h under normal culture conditions. The control group was cultured normally for the same duration.

[0104] 2.2 Cell Experiment Grouping and Intervention Methods

[0105] 2.2.1 CLG promotes NSC proliferation after OGD / R

[0106] Primary NSCs were digested into single-cell suspensions and seeded into PDL-coated 96-well or 6-well plates (2 × 10⁶ cells / well). 5Cells were cultured for 24 h with EGFR inhibitor AG1478 (10 μM). NSCs were divided into Control, OGD / R, CLG (12.5 μM), and CLG+AG1478 groups. The CLG+AG1478 group was pretreated with the EGFR inhibitor AG1478 (10 μM) for 1 h. After OGD / R treatment, upon reoxygenation, the CLG and CLG+AG1478 groups were cultured with the corresponding CLG for another 1 h, while the Control group was treated with the corresponding solvent for the same duration. Cells were then collected, and total protein was extracted. Upon reoxygenation, BrdU (10 μM) was added and the cells were cultured for 24 h for immunofluorescence assays to detect cell proliferation.

[0107] 2.2.2 FMNT promotes NSC migration after OGD / R

[0108] Neurospheres (NSCs) were digested into spheres approximately 200 μm in diameter and seeded in PDL-coated 48-well plates for overnight culture to allow for neurosphere attachment. 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. Upon reoxygenation, the FMNT group and the FMNT+SB-3CT group were cultured with FMNT (100 μM) for 24 h.

[0109] 2.2.3 AS-IV promotes NSC differentiation after OGD / R

[0110] Primary NSCs were digested into single-cell suspensions and seeded into PDL-coated 96-well or 6-well plates (2 × 10⁶ cells / well). 5 After culturing for 24 hours, NSCs were divided into Control group, OGD / R group, AS-IV group (25 μM), and AS-IV+T0070907 group. The AS-IV+T0070907 group was pretreated with T0070907 (15 μM) for 30 min. During reoxygenation, complete medium containing AS-IV or T0070907 was added. After 24 h of reoxygenation, the medium was changed to differentiation medium containing AS-IV, and cultured for another 7 days for subsequent experiments.

[0111] 3 Experimental Results

[0112] 3.1 CLG promotes NSC proliferation after OGD / R by upregulating EGFR phosphorylation levels

[0113] The results suggest that CLG concentration-dependently promotes EGFR phosphorylation, with 12.5 μM showing the best effect. Figure 8 A). Pretreatment with AG-1478 (10 μM) significantly reversed CLG-induced phosphorylation of EGFR. Figure 8B). Immunofluorescence results showed that 12.5 μM CLG significantly increased BrdU after OGD / R. + / Nestin + The proportion of cells, and AG-1478 pretreatment significantly reversed the effect of CLG ( Figure 9 ).

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

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

[0116] 3.3 AS-IV promotes neuronal differentiation of NSCs after OGD / R by upregulating PPARγ.

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

[0118] In summary, this multi-component traditional Chinese medicine formula significantly reduces neurological deficits, decreases the area of ​​cerebral infarction, promotes post-stroke neurogenesis, and improves the prognosis of cerebral infarction through multiple pathways and targets.

[0119] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. The application of a composition of active ingredients from traditional Chinese medicine in the preparation of a drug for treating cerebral infarction, characterized in that, The active ingredient composition of the traditional Chinese medicine consists of verbascoside, cymenein, and astragaloside A. The dosage of verbascoside in the medicine is 21-33 mg / kg per day, the dosage of cymenein is 6.4-25.6 mg / kg per day, and the dosage of astragaloside A is 20-60 mg / kg per day.

2. The application according to claim 1, characterized in that, The dosage forms of the drug are powder injection, tablets, capsules, powder, pills, oral liquid, ointment, and granules.

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