Pharmaceutical composition for preventing or treating ischemic encephalopathy and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a pharmaceutical composition for preventing or treating ischemic encephalopathy and application thereof, and the pharmaceutical composition comprises ginsenoside Rg1, ruscogenin and schizandrin. In an in-vitro oxygen-sugar deficiency / reperfusion induced bEnd.3 cell injury model, compared with a corresponding monomer component, the pharmaceutical composition disclosed by the invention can obviously recover the cell activity. In an in-vivo rat middle cerebral artery obstruction reperfusion model, the pharmaceutical composition disclosed by the invention can be used for remarkably reducing the cerebral infarction volume, improving the neural behavior function and recovering cerebral blood flow in an ischemic area. Meanwhile, toxicity is not shown in the continuous administration period. The results show that the pharmaceutical composition has an application prospect in treatment of cerebral arterial thrombosis, especially in improvement of ischemia reperfusion prognosis by restoring blood flow.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a pharmaceutical composition for the prevention or treatment of ischemic encephalopathy and its uses. Background Technology

[0002] Stroke, the second leading cause of death and the third leading cause of disability worldwide, seriously endangers human health. Ischemic stroke (IS) is the main type of stroke, accounting for approximately 80% of clinical cases. Currently, clinical treatment mainly focuses on thrombolysis and revascularization, but the treatment window is narrow, and ischemia-reperfusion injury (I / R) caused by the restoration of blood flow severely affects patient prognosis and increases the difficulty of treatment.

[0003] Traditional Chinese medicine is an important cradle for drug development, providing active molecules for the development of innovative drugs. Ginsenoside Rg1 (Gin), Ruscogenin (Rus), and Schisandrin A (SchA) are naturally derived active small molecules. Previous pharmacological studies have reported that they all possess neuroprotective activity. Gin belongs to the dammarane-type tetracyclic triterpenoid saponins. It is mainly derived from the roots of plants such as ginseng, Panax notoginseng, and American ginseng. It is an important quality control standard in the pharmacopoeia. Gin can activate nerve growth factor, and has antioxidant, anti-inflammatory, anti-apoptotic, and immunostimulatory activities, as well as inhibit excitotoxicity and excess calcium. 2+It plays a neuroprotective role by flowing into neurons, maintaining cellular ATP levels, and protecting the structural integrity of neurons [Yang S, Wang J, Cheng P, et al. Ginsenoside Rg1 in neurological diseases: From bench to bedside[J]. Acta Pharmacologica Sinica, 2023, 44(5):913-930.]. Rus belongs to the spirostanol type of steroidal saponin. It is mainly derived from plants such as Ophiopogon japonicus and Lily of the Valley. Ruscogenin attenuates cerebral ischemia-induced blood-brain barrier dysfunction by suppressing the interaction between NLRP3 and TXNIP proteins, thereby inhibiting inflammasome activation and the MAPK pathway and reducing the generation of reactive oxygen species (ROS). It exhibits significant anti-inflammatory and neuroprotective activities [Cao G, Jiang N, Hu Y, et al. Ruscogenin attenuates cerebral ischemia-induced blood-brain barrier dysfunction by suppressing TXNIP / NLRP3 inflammasome activation and the MAPK pathway[J]. International journal of molecular sciences, 2016, 17(9): 1418.]. SchA, a biphenylcyclooctene lignan derived from Schisandra chinensis, is its unique marker active ingredient and also meets the quality control standards stipulated in the pharmacopoeia. Schisandrin A (Schisandrin A) exerts a neuroprotective effect during cerebral ischemia by inhibiting neuroinflammation, reducing neuronal apoptosis, and regulating neurotransmitter release [Fu K, Zhou H, Wang C et al. A review: Pharmacology and pharmacokinetics of Schisandrin A. Phytother Res. 2022 Jun;36(6):2375-2393.]. Currently, single active ingredients often have inherent limitations such as a narrow therapeutic window (i.e., the toxic dose is close to the effective dose), low bioavailability, and excessively high effective doses required to achieve the expected pharmacological effect. Research based on combinations of natural active ingredients is an important direction for innovative drug development.

[0004] The chemical structures of ginsenoside Rg1 (Gin), ruscogenin (Rus), and schisandrin A (SchA) are as follows: Figure 1As shown. A search revealed no reports of an active ingredient composition (ACCS) consisting of Gin, Rus, and SchA in the treatment of ischemic stroke, particularly in terms of restoring blood flow and improving the ischemia-reperfusion pathology. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention provides a pharmaceutical composition for the prevention or treatment of ischemic encephalopathy and its use.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a pharmaceutical composition (ACCS) for the prevention or treatment of ischemic encephalopathy, the pharmaceutical composition comprising ginsenoside Rg1, ruscosaponin and schisandrin A.

[0007] Alternatively, in the above pharmaceutical composition, the mass ratio of ginsenoside Rg1, ruscosaponin and schisandrin is in the range of (2-8):(1-5):(1-5).

[0008] Alternatively, in the above pharmaceutical composition, the mass ratio of ginsenoside Rg1, ruscosaponin and schisandrin A is 5:3:2.

[0009] In a second aspect, the present invention provides a pharmaceutical formulation comprising the pharmaceutical composition described in the first aspect above, and one or more pharmaceutically acceptable excipients.

[0010] Preferably, the pharmaceutically acceptable excipients of the present invention include conventional pharmaceutical solvents (such as water, ethanol, propylene glycol, injectable oils, etc.), diluents (such as starch, powdered sugar, dextrin, lactose, pregelatinized starch, microcrystalline cellulose, inorganic calcium salts (such as calcium sulfate, calcium hydrogen phosphate, pharmaceutical-grade calcium carbonate, etc.), mannitol, vegetable oils, polyethylene glycol, etc.), binders (such as water, ethanol, starch paste, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose and ethyl cellulose, hydroxypropyl methyl cellulose, etc.), and disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted...). Hydroxypropyl cellulose, croscarmellose, croscarmellose sodium carboxymethyl cellulose, etc.), lubricants (such as magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol, magnesium lauryl sulfate, etc.), absorption enhancers (such as surfactants, Azone, EDTA, salicylic acid, amino acid ethylamine derivatives, acetoacetates, β-dicarboxylic acid esters, aromatic acidic compounds, aliphatic acids, etc.), preservatives (such as benzoic acid, hydroxypropyl butyl ester, hydroxypropyl methyl ester, phenol, m-cresol, etc.), flavoring agents (such as sucrose, stevioside, etc.), etc.

[0011] Alternatively, in the above-mentioned pharmaceutical preparations, the pharmaceutical preparations may be oral or non-oral preparations.

[0012] Alternatively, in the above-mentioned pharmaceutical preparations, the oral preparation is selected from tablets, capsules, pellets, granules, powders, oral films, or oral liquids.

[0013] Alternatively, in the above-mentioned pharmaceutical preparations, the non-oral preparation is selected from injections, ointments, creams, or suppositories.

[0014] In a third aspect, the present invention provides the use of the pharmaceutical composition described in the first aspect or the pharmaceutical preparation described in the second aspect in the preparation of a medicament for the prevention or treatment of ischemic encephalopathy.

[0015] Alternatively, in the above-described uses, the ischemic encephalopathy is ischemic stroke.

[0016] Alternatively, in the above-described uses, the ischemic stroke is selected from focal ischemic stroke or diffuse ischemic stroke.

[0017] Alternatively, in the above-mentioned uses, the drug can restore cerebral blood flow and improve the pathological process of cerebral ischemia-reperfusion.

[0018] When used to treat ischemic stroke, the drug of the present invention is administered to humans or mammals. For this purpose, preferably, the mass of the drug of the present invention ingested or administered, based on a ginsenoside Rg1:ruscosaponin:schisandrin mass ratio of 5:3:2, is 50-400 mg per adult body weight of 60-70 kg, more preferably 100-200 mg per person per day.

[0019] Compared with the prior art, the present invention has the following advantages: This invention provides a novel natural product active ingredient composition (ACCS) for the prevention or treatment of ischemic encephalopathy. Pharmacological tests show that in an in vitro oxygen-glucose deficiency / reperfusion (OGD / R)-induced bEnd.3 cell injury model, ACCS significantly restores cell viability compared to the corresponding monomeric components. In an in vivo rat middle cerebral artery occlusion (MCAO) reperfusion model, ACCS significantly reduces infarct volume, improves neurobehavioral function, and restores cerebral blood flow to the ischemic area. Furthermore, ACCS can restore neuronal structure in the ischemic area, increase the number of Nissl bodies and MAP2-positive cells, and its activity is equivalent to that of the positive control drug nimodipine (Nim). No toxicity was observed during continuous administration. These results demonstrate the application potential of ACCS in the treatment of ischemic stroke, particularly by restoring blood flow and improving the prognosis of ischemia-reperfusion. Attached Figure Description

[0020] The present invention will be further described below with reference to the accompanying drawings.

[0021] Figure 1 Chemical formulas of ginsenoside Rg1, ruscosaponin, and schisandrin A.

[0022] Figure 2 The results show the changes in cell viability after OGD / R modeling and administration of ACCS and its single components as detected by MTT assay. Data are presented as mean ± standard deviation (Means ± SD). # Comparison with the sham group p <0.05; ## : Compared with the control group p <0.01; Compared with the OGD / R group p <0.05; Compared with the OGD / R group p <0.01; ns: no significant difference.

[0023] Figure 3 This study demonstrated that ACCS has a protective effect against cerebral ischemia-reperfusion injury in MCAO rats. Figure 3 A is the Zea-Longa score used to assess neurological deficits. Figure 3 B represents the magnetic resonance imaging (MRI) findings in the infarcted area. Scale bar: 2000 μm. Figure 3 C represents the quantitative comparison of cerebral blood flow between ischemic and non-ischemic areas using laser speckle contrast imaging. Scale bar: 2000 μm. Data are expressed as mean ± standard deviation (Means ± SD). # Comparison with the sham group p <0.05; ## Comparison with the sham group p <0.01; Compared with the MCAO group p <0.05; Compared with the MCAO group p <0.01; ns: no significant difference.

[0024] Figure 4 ACCS was shown to reduce neuronal damage in MCAO rats without significant toxicity. Figure 4 Image A shows representative images of Nissl staining and hematoxylin-eosin staining, revealing the morphology and structural integrity of neurons in brain tissue. Scale bar: 50 μm. Figure 4 B is an immunohistochemical analysis of microtubule-associated protein 2 expression in the cerebral infarction region. Scale bar: 50 μm. Figure 4C represents the histopathological evaluation of major organs (heart, liver, spleen, lung, and kidney) after hematoxylin-eosin staining. Scale bar: 50 μm. Detailed Implementation

[0025] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products. The purchase details of some raw materials and reagents are as follows: Ginsenoside Rg1, Ruscosaponin, and Schisandrin A were purchased from Chengdu Desite Biotechnology Co., Ltd. Their structures were determined by UV, MS, NMR and other methods, and their purity was above 98%.

[0027] Brain microvascular endothelial cells bEnd.3 were purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0028] DMEM culture medium was purchased from Thermo Fisher Scientific, USA. Fetal bovine serum (FBS), penicillin-streptomycin antibiotics, dimethyl sulfoxide (DMSO), nimodipine (Nim), and carboxymethyl cellulose sodium (CMC-Na) were purchased from Sigma-Aldrich, USA. 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Shanghai Taosu Biochemical Technology Co., Ltd. EBSS buffer was purchased from Beijing Solarbio Science & Technology Co., Ltd. Isoflurane and suture plugs were purchased from Shenzhen Ruiward Life Science & Technology Co., Ltd. All other reagents were of analytical grade and purchased from a Beijing chemical plant.

[0029] Example 1: ACCS restores OGD / R-induced bEnd.3 cell viability damage. 1.1 Test Cells bEnd.3 cells were cultured in DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics. Cells were cultured in a constant temperature and humidity incubator at 37 °C containing 5% carbon dioxide (CO2).

[0030] 1.2 Test Drug Accurately weigh ginsenoside Rg1, ruscosaponin, and schisandrin A, and dissolve them separately in DMSO to prepare 20 mM stock solutions. Aliquots and store at -20 °C. Dissolve ACCS (ginsenoside Rg1, ruscosaponin, and schisandrin A in a mass ratio of 5:3:2) in DMSO to prepare a 20 mM stock solution, and aliquot and store at -20 °C. Dilute the stock solutions to complete culture medium before experimentation.

[0031] 1.3 Establishment of Oxygen-Glucose Deprivation / Reperfusion (OGD / R) Model and Drug Administration Cells in the logarithmic growth phase were seeded into 96-well plates. When the cells reached 50-60% confluence, the complete culture medium was discarded, and the cells were gently washed twice with pre-warmed EBSS balanced salt solution. 100 μL of EBSS solution was added to each well. The 96-well plates were transferred to a tri-gas incubator and cultured at 37 °C for 6 h. After incubation, the cells were removed from the hypoxic chamber, replaced with normal culture medium, and reperfused in a 37 °C, 5% CO2 cell culture incubator for another 24 h. The treatment groups received ginsenoside Rg1 (20 μM), ruscosaponin (20 μM), schisandrin A (20 μM), and ACCS (20 μM), respectively. The control and model groups received an equal volume of DMSO.

[0032] 1.4 MTT assay for cell viability After drug intervention, the old culture medium was discarded, and MTT working solution with a final concentration of 0.5 mg / mL was added. The cells were incubated at 37 °C in the dark for 4 h. The MTT solution in the wells was carefully aspirated, and DMSO was added to dissolve the formazan generated during the reaction. The absorbance (A) was then measured at 570 nm using a microplate reader. Cell viability = (A... 实验孔 -A 空白孔 ) / (A 对照孔 -A 空白孔 ).

[0033] 1.5 Test Results bEnd.3 cell viability was detected using the MTT assay. Figure 2 As shown, the results indicated that after OGD / R modeling, cell viability decreased to approximately 50% of normal cells. After ACCS (20 μM) intervention, cell viability significantly recovered. Further comparison revealed that neither ginsenoside Rg1 (20 μM), ruscosaponin (20 μM), nor schisandrin A (20 μM) alone significantly reversed OGD / R-induced cell damage. These results suggest that ACCS has a superior repair effect on OGD / R-induced cell damage compared to its individual components, indicating that ACCS may exert a more significant cytoprotective effect through a multi-component synergistic mechanism.

[0034] The results of this embodiment are as follows: ACCS can restore the viability of OGD / R-induced bEnd.3 cells, and its effect is superior to that of any single component.

[0035] Example 2: ACCS has a protective effect against cerebral ischemia-reperfusion injury in MCAO rats. 1.1 Test animals Sprague-Dawley (SD) rats, 6-8 weeks old, male, purchased from the Experimental Animal Center of Peking University.

[0036] 1.2 Test Drug Ginsenoside Rg1, ruscosaponin, and schisandrin A were mixed in a mass ratio of 5:3:2 to prepare ACCS. ACCS was dissolved in 5% CMC-Na solution, and the dosage was 20 mg / kg. The positive control drug nimodipine (Nim) was dissolved in 5% CMC-Na solution, and the dosage was 20 mg / kg. All drugs were administered intraperitoneally once daily for 7 consecutive days. The sham-operated group and the model group received an equal volume of 5% CMC-Na solution.

[0037] 1.3 Middle Cerebral Artery Occlusion (MCAO) Reperfusion Model Rats were randomly divided into four groups: sham-operated group (Sham group), MCAO model group, MCAO + ACCS group, and MCAO + Nim group. The MCAO model was established as follows: Under isoflurane anesthesia, the right common carotid artery, external carotid artery, and internal carotid artery were isolated; after ligating the external carotid artery, a suture was inserted through the common carotid artery to the internal carotid artery; the suture was advanced approximately 18-20 mm to occlude the right middle cerebral artery. The suture was withdrawn 1.5 h after occlusion to allow reperfusion. Anesthesia was maintained continuously with isoflurane during the procedure, and body temperature was maintained at 37 °C using a heating pad.

[0038] 1.4 Neurological deficit score The degree of neurological deficit was assessed using the Zea-Longa scoring system: 0 points, normal, no neurological deficit; 1 point, mild deficit, left forelimb cannot be fully extended; 2 points, moderate deficit, turning to the left when walking; 3 points, severe deficit, falling to the left; 4 points, no spontaneous activity, decreased consciousness; 5 points, death.

[0039] 1.5 Magnetic Resonance Imaging (MRI) Following the last administration, rats were anesthetized and fixed in a supine position on the scanning bed, with their heads placed within a dedicated experimental coil (40 mm in diameter). Scanning was performed using a GE Healthcare magnetic resonance imaging system. T2-weighted imaging employed a fast spin-echo sequence with the following parameters: repetition time 3767 ms, echo time 55.3 ms, field of view 4 cm, matrix 129 × 256, one excitation, 11 consecutive scans, and a slice thickness of 1.5 mm. The coronal plane displaying the largest infarct area was selected for quantitative assessment of the infarct area: infarct volume (%) = (infarct volume / total brain volume) × 100%.

[0040] 1.6 Laser Speckle Contrast Imaging (LSCI) Rats were fixed in a stereotactic frame, and a cranial window (10 mm × 15 mm) was prepared. Cerebral blood flow (CBF) was monitored using a laser speckle contrast imaging system equipped with a 785 nm laser source and a complementary metal-oxide-semiconductor (CMOS) camera at a frame rate of 100 fps and a spatial resolution of 7.5 million points / cm². The probe was positioned approximately 20 cm above the skull surface, and the camera resolution was set to 2064 × 1544 pixels. The raw speckle images were processed using system software to generate perfusion maps. The cerebral blood flow perfusion in the infarcted area was quantitatively analyzed by comparing the average signal intensity between the ischemic and non-ischemic areas. Perfusion volume (%) = (CBF) / (Cerebral blood flow perfusion rate). 缺血区 / CBF 非缺血区 ) × 100%.

[0041] 1.7 Test Results A rat model of cerebral ischemia-reperfusion injury (MCAO) was established, and the in vivo anti-cerebral ischemia-reperfusion efficacy of ACCS (20 mg / kg) and the positive control drug Nim (20 mg / kg) were evaluated. Neurological deficit assessment using the Zea-Longa scoring system revealed that, compared to the sham group, the MCAO group exhibited significant neurological deficits, specifically abnormal behaviors such as crossed paws, circling, and lateral tilting; both ACCS and Nim interventions significantly improved these neurological deficits. Figure 3 A). Further magnetic resonance imaging (MRI) revealed significantly high signal intensity in the ischemic lateral brain region of the MCAO group rats, suggesting cerebral infarction; in contrast, the infarct volume was significantly reduced in the ACCS and Nim groups. Figure 3 B). Furthermore, LSCI results showed that cerebral blood flow (CBF) perfusion was significantly reduced and local vascular density decreased after MCAO; after ACCS and Nim treatment, cerebral blood flow perfusion and vascular abundance were effectively restored, and the ischemic state was significantly improved. Figure 3 C). The above results indicate that ACCS can effectively improve neurological deficits, reduce the extent of cerebral infarction, and promote the recovery of cerebral blood flow in MCAO rats, exhibiting neuroprotective effects comparable to those of the positive control drug Nim.

[0042] The results of this embodiment are as follows: 1) ACCS can effectively improve neurological function damage in MCAO rats.

[0043] 2) ACCS can effectively reduce the cerebral infarction area and restore cerebral blood flow in the ischemic area of ​​MCAO rats.

[0044] 3) ACCS has a protective effect against cerebral ischemia-reperfusion injury in MCAO rats that is comparable to that of the positive control drug Nim.

[0045] Example 3: ACCS has a protective effect against cerebral ischemia-reperfusion injury in MCAO rats. 1.1 Test animals Sprague-Dawley (SD) rats, 6-8 weeks old, male, purchased from the Experimental Animal Center of Peking University.

[0046] 1.2 Test Drug Ginsenoside Rg1, ruscosaponin, and schisandrin A were mixed in a mass ratio of 5:3:2 to prepare ACCS. ACCS was dissolved in 5% CMC-Na solution, and the dosage was 20 mg / kg. The positive control drug nimodipine (Nim) was dissolved in 5% CMC-Na solution, and the dosage was 20 mg / kg. All drugs were administered intraperitoneally once daily for 7 consecutive days. The sham-operated group and the model group received an equal volume of 5% CMC-Na solution.

[0047] 1.3 Middle Cerebral Artery Occlusion (MCAO) Reperfusion Model Rats were randomly divided into four groups: sham-operated group (Sham group), MCAO model group, MCAO+ACCS group, and MCAO+Nim group. The MCAO model was established as follows: Under isoflurane anesthesia, the right common carotid artery, external carotid artery, and internal carotid artery were isolated; after ligating the external carotid artery, a suture was inserted through the common carotid artery to the internal carotid artery; it was gently advanced to a depth of approximately 18–20 mm to occlude the right middle cerebral artery. The suture was withdrawn 1.5 h after occlusion to allow reperfusion. Anesthesia was maintained continuously with isoflurane during the procedure, and body temperature was maintained at 37 °C using a heating pad.

[0048] 1.4 Hematoxylin and eosin (H&E) staining After the last administration, rats were euthanized due to overdose anesthesia. Brain tissue was obtained, followed by heart, liver, spleen, lung, and kidney tissues. The tissues were fixed with 4% paraformaldehyde for 24 h. The tissues were dehydrated using a gradient of ethanol, treated with 50%, 70%, 85%, and 95% ethanol for 90 min each, then with 100% ethanol for 60 min, repeated once. The dehydrated tissues were then immersed in xylene for 90 min, followed by paraffin embedding. The embedded tissue blocks were placed on a microtome and cut into 5 μm thick coronal sections, which were then spread in warm water and attached to glass slides, and baked at 60 °C for 5 h. The sections were then dewaxed in xylene for 10 min, repeated once. They were then immersed in anhydrous ethanol, 95%, 90%, 80%, 70%, and distilled water for 10 min each. The sections were stained with hematoxylin and eosin, respectively, and washed with water to remove unbound staining solutions. The stained sections were then dehydrated using a gradient of ethanol, permeated with xylene, and mounted with neutral resin. Imaging was performed using a digital pathology slide scanner, with the mode set to bright field and observed at 40 ×. Images were processed using NDP.view2 software.

[0049] 1.5 Nissl staining Brain tissue paraffin sections were obtained according to 1.4, and then dewaxed and hydrated. Nissl stain was applied at 37 °C for 30 min. The stain was removed by washing with water. Dehydration, permeabilization, and mounting were performed as in 1.4. Images were imaged using a digital pathology scanner, with the mode set to bright field and observed at 40×. Images were processed using NDP.view2 software.

[0050] 1.6 Immunohistochemistry (IHC) Brain tissue paraffin sections were obtained according to 1.4. The sections were immersed in dewaxing antigen retrieval solution and subjected to antigen retrieval using an automated dewaxing and hydration staining system. The program was set to high heat for 5 min, low heat for 20 min, and then allowed to cool naturally to room temperature. The sections were rinsed three times with PBS to remove residual retrieval solution. The tissue area was circled with an immunohistochemical pen, and the sections were placed in a humidified chamber. They were then incubated with 3% methanol-H2O2 in the dark for 10 min to block endogenous peroxidase. Subsequently, they were blocked with 5% goat serum at room temperature for 30 min to block non-specific binding sites. After removing the blocking solution, a suitable amount of diluted primary antibody was added and incubated overnight at 4 °C. The sections were rinsed three times with PBS, and a secondary antibody corresponding to the primary antibody species was added, incubating at room temperature for 1 h. The sections were rinsed three times with PBS, and DBA chromogenic solution was added, incubating in the dark until sufficient color development was achieved. Subsequent dehydration, permeabilization, and mounting procedures were the same as in 1.4. The sections were imaged using a digital pathology slide scanner, set to bright field mode, and observed at 40 ×. Images were processed using NDP.view2 software.

[0051] 1.7 Test Results Nissl staining and hematoxylin-eosin staining were used to observe neuronal damage in the infarct area. In the sham-operated group, neurons were neatly arranged, morphologically intact, and rich in Nissl bodies. Figure 4 A). In contrast, the MCAO model group exhibited severe histological damage, including neuronal structural disorder, significant edema and necrosis, and a significant reduction in the number of Nissl bodies. After intervention with ACCS and Nim, the above pathological changes were significantly improved, and the brain tissue structure and cell morphology were basically restored.

[0052] Microtubule-associated protein 2 (MAP2) is a key cytoskeletal protein that maintains neuronal morphology and neurite growth. We further conducted immunohistochemical analysis. The results showed that the number of MAP2-positive cells was significantly reduced in the MCAO group, while both ACCS and Nim treatments increased the number of MAP2-positive cells. Figure 4 B). Furthermore, H&E staining of major organs (heart, liver, spleen, lungs, kidneys) indicated that ACCS administration did not cause significant pathological changes in major organs. Figure 4 C). In summary, the results indicate that ACCS can effectively reduce neuronal death in the infarct area, and no obvious toxic reactions were observed.

[0053] The results of this embodiment are as follows: 1) ACCS can significantly improve the structure and restore cell morphology of ischemic brain tissue in MCAO rats.

[0054] 2) ACCS can increase the number of MAP2 positive cells in the ischemic area of ​​MCAO rats.

[0055] 3) ACCS showed no significant organ toxicity with continuous administration.

[0056] Obviously, the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the invention. Therefore, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention also intends to include these modifications and variations.

Claims

1. A pharmaceutical composition for the prevention or treatment of ischemic encephalopathy, characterized in that: The pharmaceutical composition comprises ginsenoside Rg1, ruscosaponin, and schisandrin A.

2. The pharmaceutical composition according to claim 1, characterized in that: In the pharmaceutical composition, the mass ratio of ginsenoside Rg1, ruscosaponin and schisandrin ranges from (2-8):(1-5):(1-5).

3. The pharmaceutical composition according to claim 2, characterized in that: In the pharmaceutical composition, the mass ratio of ginsenoside Rg1, ruscosaponin and schisandrin A is 5:3:

2.

4. A pharmaceutical preparation, characterized in that: The pharmaceutical preparation comprises the pharmaceutical composition of any one of claims 1 to 3, and one or more pharmaceutically acceptable excipients.

5. The pharmaceutical preparation according to claim 4, characterized in that: The pharmaceutical preparation is an oral or non-oral preparation.

6. The pharmaceutical preparation according to claim 5, characterized in that: The oral preparations are selected from tablets, capsules, pellets, granules, powders, oral films, or oral liquids.

7. The pharmaceutical preparation according to claim 5, characterized in that: The non-oral preparations are selected from injections, ointments, creams, or suppositories.

8. Use of the pharmaceutical composition of any one of claims 1 to 3 or the pharmaceutical preparation of any one of claims 4 to 7 in the preparation of a medicament for the prevention or treatment of ischemic encephalopathy.

9. The use according to claim 8, characterized in that: The ischemic encephalopathy mentioned is ischemic stroke.

10. The use according to claim 9, characterized in that: The ischemic stroke is selected from focal ischemic stroke or diffuse ischemic stroke.

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