Pharmaceutical composition for treating ischemic stroke

Abstract

The invention provides a medicine composition for treating ischemic stroke, and the medicine composition comprises the following active ingredients: chlorogenic acid, loganin, albiflorin, paeoniflorin, parishin A, cinnamic acid, beta-asarone and isoimperatorin. Clinically, the pharmaceutical composition can remarkably improve the serum oxidative stress level and the neurological function impairment symptom of a patient with cerebral infarction in the acute stage, improves the life quality of the patient, meanwhile, proves that the pharmaceutical composition can inhibit ferroptosis by adjusting a GPX4 / ACSL4 / pathway in fundamental research, effectively improves mitochondrial structure and function impairment, and has a good clinical application prospect. A new treatment thought is provided for treating ischemic stroke.

Description

Technical Field

[0001] The present invention relates to the technical field of drug combinations, and more specifically, to a drug composition for treating ischemic stroke. Background Art

[0002] Cerebral infarction, also known as stroke in traditional Chinese medicine, accounts for a high proportion of cerebrovascular diseases and can occur at any age. Patients with cerebral infarction generally experience partial or incomplete loss of mobility, which places a heavy burden on individuals, families, and society. Therefore, it is particularly important to study effective drugs for the treatment of cerebral infarction.

[0003] With the continuous understanding of the pathogenesis of stroke and clinical experimental verification, it is proposed that the main core pathogenesis of ischemic stroke is "toxic damage to brain collaterals". The "toxic" in "toxic damage to brain collaterals" is the accumulation of multiple pathological factors such as fire, heat, phlegm, and blood stasis. Under the influence of factors such as diet, emotions, and fatigue, the function of storage and the circulation of qi and blood are abnormal, so that the physiological or pathological products in the body cannot be discharged in time, and the accumulation of phlegm and blood stasis in the body, which leads to poison over time, obstruction of blood vessels, disorder of qi and blood, and damage to brain collaterals and brain marrow, forming a pathological evolution process of "phlegm and blood stasis generating poison, poison damaging brain collaterals, and collateral damage to marrow", which clinically manifests as stroke symptoms such as body apraxia.

[0004] The existing drugs for treating similar diseases mainly include:

[0005] Xiaoshuantongluo Tablets: promote blood circulation and dissipate blood stasis, warm the meridians and dredge the collaterals. Indications: hyperlipidemia, mental dullness caused by cerebral thrombosis, hard tongue, delayed speech, unclear pronunciation, cold hands and feet, and pain in movement. Mainly used for blood stasis, phlegm and dampness blocking the meridians.

[0006] Dahuoluo Pills: dispel wind and relieve pain, remove dampness and resolve phlegm, relax tendons and activate collaterals. Mainly used for hemiplegia caused by ischemic stroke, pain caused by rheumatism (rheumatic arthritis), venous contracture, waist and leg pain, walking difficulties caused by traumatic injuries, and chest pain and heart pain.

[0007] However, the above existing treatments are mainly for treating symptoms, focusing on "removing blood stasis, clearing the blockage" and so on, which can alleviate symptoms but are difficult to cure the root cause. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a pharmaceutical composition for treating ischemic stroke, which can significantly improve the serum oxidative stress level and neurological dysfunction symptoms in patients in the acute phase of cerebral infarction. Basic research has confirmed that the pharmaceutical composition inhibits ferroptosis by regulating the GPX4 / ACSL4 / pathway, improves mitochondrial structure and functional damage, and provides a new therapeutic idea for the treatment of ischemic stroke.

[0009] In order to achieve the above object, the technical solution of the present invention is as follows:

[0010] A pharmaceutical composition for treating ischemic stroke, wherein the active ingredients thereof include chlorogenic acid, loganin, paeoniflorin, paeoniflorin, barisenoside A, cinnamic acid, β-asarone and isoimperatorin.

[0011] In some embodiments, the pharmaceutical composition comprises, by weight, 1-5 parts of chlorogenic acid, 2-8 parts of loganin, 3-10 parts of paeoniflorin, 5-15 parts of paeoniflorin, 10-30 parts of barisenoside A, 1-5 parts of cinnamic acid, 5-15 parts of β-asarone and 1-5 parts of isoimperatorin as active ingredients.

[0012] In some embodiments, the pharmaceutical composition comprises, by weight, 2 parts of chlorogenic acid, 4 parts of loganin, 5 parts of paeoniflorin, 8 parts of paeoniflorin, 20 parts of barisenoside A, 2 parts of cinnamic acid, 8 parts of β-asarone, and 2 parts of isoimperatorin as active ingredients.

[0013] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0014] In some embodiments, the dosage form of the pharmaceutical composition includes powder, soft capsule, granule, pill, tablet, decoction and hard capsule.

[0015] In some embodiments, the amount of the excipient used is in a ratio of 1:0.95-1.05 to the total mass of the active ingredient.

[0016] In some embodiments, the auxiliary material includes at least one of a filler, a binder, a lubricant, and a solubilizer.

[0017] In some embodiments, the auxiliary material includes at least one of starch, dextrin, sucrose, milk powder, sweetener, mannitol, lactose, cyclodextrin, disodium EDTA, magnesium stearate, calcium stearate, talc, flavor and sodium lauryl sulfate.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The pharmaceutical composition of the present invention uses chlorogenic acid, loganin, paeoniflorin, paeoniflorin, parisonoside A, cinnamic acid, β-asarone and isoimperatorin as active ingredients. The ingredients in the pharmaceutical composition and their effects are as follows:

[0020] 1. Chlorogenic Acid

[0021] Efficacy: Anti-oxidation, anti-inflammatory, protects vascular endothelium, inhibits free radical production, and regulates immune response.

[0022] Mechanism of action: Alleviate ischemic damage by scavenging free radicals and regulating redox balance.

[0023] 2. Loganin

[0024] ·Effects: Neuroprotective, anti-inflammatory, antioxidant.

[0025] Mechanism of action: Inhibits the release of inflammatory mediators and reduces neuronal damage.

[0026] 3. Paeoniflorin

[0027] Efficacy: anti-inflammatory, analgesic, improve microcirculation, neuroprotective.

[0028] Mechanism of action: Protects nerve cells by inhibiting the release of inflammatory mediators and inhibiting calcium overload.

[0029] 4. Paeoniflorin

[0030] ·Efficacy: anti-inflammatory, antioxidant, sedative, protects brain tissue.

[0031] Mechanism of action: Improves mitochondrial function and reduces oxidative stress and inflammation.

[0032] 5. Balisonoside A

[0033] Efficacy: Anti-inflammatory, immunomodulatory, and anti-neurodegenerative diseases.

[0034] Mechanism of action: Regulates the immune system and alleviates damage caused by inflammatory mediators.

[0035] 6. Cinnamic Acid

[0036] Efficacy: Anti-oxidation, anti-inflammatory, improve hemodynamics.

[0037] Mechanism of action: Inhibit lipid peroxidation and inflammatory factor activity, and improve microcirculation.

[0038] 7. β-Asarone

[0039] Efficacy: Protect neurons, fight inflammation, and improve cognitive function.

[0040] Mechanism of action: Promotes neuronal survival through regulation of neurotransmitters and antioxidant effects.

[0041] 8.Isoprugine

[0042] Efficacy: Anti-oxidation, anti-inflammatory, promotes nerve regeneration.

[0043] Mechanism of action: Inhibit free radicals and inflammatory responses, and promote nerve function repair.

[0044] The synergistic effects after compatibility are as follows:

[0045] Ferroptosis inhibition: All ingredients work together to inhibit ferroptosis by regulating the GPX4 / ACSL4 pathway, protecting neurons and reducing damage to mitochondrial structure and function.

[0046] Anti-inflammatory and immunomodulatory: Through anti-inflammatory effects at multiple targets, it reduces the release of inflammatory mediators and alleviates tissue pathological damage.

[0047] Antioxidant and mitochondrial protection: Enhances the antioxidant capacity of mitochondria, improves energy metabolism, and prevents damage caused by oxidative stress.

[0048] Brain protection and neurological repair: reduce the volume of cerebral infarction, increase the number of neurons, and improve neurological deficits.

[0049] Improve blood circulation: By dilating blood vessels and regulating microcirculation, it promotes the recovery of cerebral blood flow and reduces ischemic damage. The ingredients are reasonably matched. Clinical studies have shown that the drug composition can significantly improve the serum oxidative stress level and neurological deficit symptoms in patients with acute cerebral infarction. Basic research has confirmed that it can inhibit ferroptosis by regulating the GPX4 / ACSL4 / pathway, effectively improve mitochondrial structure and functional damage, and at the same time reduce the infarction volume, reduce pathological damage, increase the number of neurons, improve the symptoms of neurological deficit after ischemic stroke, and provide a new idea for the treatment of ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The infarct area of ​​brain cell tissue in each group of rats;

[0051] Figure 2 Figure 2 shows the pathological damage of brain tissues of rats in each group (×400 times);

[0052] Figure 3 The ultrastructure of mitochondria in neurons of brain tissue of rats in each group;

[0053] Figure 4 The expression of GPX4 and ACSL4 in the hippocampus of brain tissue of rats in each group;

[0054] Figure 5 This is the expression mark of barbed wire-related proteins GPX4 and ACSL4. DETAILED DESCRIPTION

[0055] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0057] Example 1

[0058] 1. Main drugs and reagents

[0059] Huoxuetongluo Pills: The active ingredients are 30mg of honeysuckle vine, 30mg of Photinia serrulata, 10mg of peach kernel, 10mg of Vaccaria segetalis, 15mg of Gastrodia elata, 15mg of Scrophularia ningpoensis, 10mg of Paeonia lactiflora, 15mg of Papaya, 10mg of Acorus calamus, 10mg of Angelica dahurica, 6mg of Gleditsia sinensis, 10mg of Gentiana macrophylla, and 0.3mg of Borneol. Provided by the Department of Pharmaceutical Preparation of the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine, the conventional dosage for patients with cerebral infarction is 15g / d.

[0060] Huoxuerongluo component tablets: component combination tablets, composed of 2mg of isoimperatorin, 4mg of chlorogenic acid, 20mg of barleyson glycoside A, 2mg of cinnamic acid, 8mg of paeoniflorin, 5mg of paeoniflorin, 4mg of loganin and 8mg of β-asarone, totaling 51mg, in the form of cyclodextrin inclusion compound. Huoxuerongluo component tablets with a total tablet weight of 101mg are prepared by pretreatment of active ingredients, dry mixing, wet granulation, drying and tableting. The conventional dosage for patients with cerebral infarction is 9 tablets / d (909mg / d).

[0061] 2. Information

[0062] A total of 120 patients with acute cerebral infarction admitted to the Department of Neurology of the First Affiliated Hospital of Hunan University of Chinese Medicine from January 2022 to January 2024 were selected for the study and divided into three groups with 40 cases in each group using the random number table method. Inclusion criteria: 1. Meet the diagnostic criteria of cerebral infarction in the "Guidelines for the Diagnosis and Treatment of Cerebral Infarction in China with Integrated Traditional Chinese and Western Medicine (2017)", 2. Confirmed by imaging examination and all are first-time onset, 3. Complete clinical data. Exclusion criteria: 1. Patients with dysfunction of important organs such as liver and kidney, 2. Patients with hemorrhagic diseases, including hemorrhagic stroke (cerebral hemorrhage, subarachnoid hemorrhage, etc.), gastrointestinal bleeding, severe coagulation dysfunction, etc., 3. Patients with malignant tumors or mental diseases, 4. Patients who have received other traditional Chinese medicine treatments within the past month, 5. Patients who are allergic to the drugs used in this study. This study was approved by the Ethics Committee, and the patients or their families were informed and signed the consent form. There was no statistically significant difference in the baseline data between the two groups (P>0.05). See Table 1 for details.

[0063] Table 1 Comparison of general information of patients in the three groups

[0064]

[0065] 3. Method

[0066] The control group was given conventional treatments such as antiplatelet aggregation, lipid-lowering and plaque-stabilizing, neuroprotection, blood pressure and blood glucose control, etc., and additionally 25 mg of butylphthalide sodium chloride injection was added, twice a day, all provided by the Central Pharmacy and the Intravenous Admixture Service Center of the First Affiliated Hospital of Hunan University of Chinese Medicine.

[0067] The Huoxue Rongluo Pills group was given Huoxue Rongluo Pills on the basis of the control group, 5 g, three times a day, provided by the In-hospital Preparation Center of the First Affiliated Hospital of Hunan University of Chinese Medicine.

[0068] The Huoxue Rongluo Tablets group was given Huoxue Rongluo Component Tablets (the combination of components of the present invention) on the basis of the control group, 3 tablets, three times a day.

[0069] All three groups were treated continuously for 14 days.

[0070] 4. Observation Indicators

[0071] Clinical efficacy: The clinical efficacy of the three groups of patients after treatment was evaluated according to the National Institutes of Health Stroke Scale (NIHSS). A score reduction of 0-17%, 18%-44%, 45%-89%, and 90%-100% represented ineffective, improved, significantly improved, and cured, respectively. The total effective rate = (number of cured + significantly improved + improved) cases / total number of cases × 100%. Activities of daily living: The BI was used for evaluation. A BI score ≤ 20 points indicated complete need for help; 20 points < BI score ≤ 40 points indicated more need for help; 40 points < BI score ≤ 60 points indicated need for help; BI score > 60 points indicated basically able to complete by oneself. Serum oxidative stress indicators included malondialdehyde (MDA), serum glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD). Approximately 5 ml of fasting venous blood was collected from the patients in the early morning, and it was detected by the Clinical Laboratory of the First Affiliated Hospital of Hunan University of Chinese Medicine.

[0072] 5. Results

[0073] 5.1 Comparison of NHISS scores and BI index scores

[0074] Before treatment, there was no significant difference in NHISS scores and BI index scores among the three groups (P>0.05). After treatment, NHISS scores of the three groups were lower than those before treatment, neurological deficits were improved, BI index scores of the three groups were higher than those before treatment, and daily living ability was improved. After treatment, the NHISS scores of Huoxue Rongluo Pills and Huoxue Rongluo Group Tablets were lower than those of the control group, and the NHISS scores of Huoxue Rongluo Group Tablets were lower than those of the control group. The BI index scores of Huoxue Rongluo Pills and Huoxue Rongluo Group Tablets were higher than those of the control group, and the BI index score of Huoxue Rongluo Pills was higher than that of the control group, and the BI index score of Huoxue Rongluo Group Tablets was higher than that of the control group. The differences were all statistically significant (P<0.05), as shown in Table 2.

[0075] Table 2 Comparison of NHISS scores and BI index scores among the three groups of patients before and after treatment ( point)

[0076]

[0077]

[0078] Note: Compared with the control group after treatment, * P<0.05; compared with the Huoxue Rongluo Pills group after treatment, ▲ P<0.05; compared with the group before treatment, # P<0.05.

[0079] 5.2 Comparison of clinical efficacy

[0080] The total effective rate of the Huoxue Rongluo tablet group was 100%, which was higher than 85% of the Huoxue Rongluo pill group and 60% of the control group. The differences were statistically significant (P < 0.05), as shown in Table 3.

[0081] Table 3 Comparison of clinical efficacy among three groups of patients

[0082]

[0083] Note: Compared with the control group after treatment, * P<0.05; compared with the treatment of Huoxue Rongluo Pills, ▲ P<0.05

[0084] 5.3 Comparison of serum oxidative stress levels

[0085] Before treatment, there were no significant differences in MDA, SOD and GSH-Px among the three groups (P>0.05). After treatment, the MDA levels of the three groups were reduced, and the MDA levels of the Huoxue Rongluo Pills group and the Huoxue Rongluo Tablets group were lower than those of the control group, and the MDA level of the Huoxue Rongluo Tablets group was lower than that of the Huoxue Rongluo Pills group. The activities of SOD and GSH-Px in the three groups were increased, and the activities of SOD and GSH-Px in the Huoxue Rongluo Pills group and the Huoxue Rongluo Tablets group were higher than those in the control group, and the activities of SOD and GSH-Px in the Huoxue Rongluo Tablets group were higher than those in the Huoxue Rongluo Pills group, and the differences were statistically significant (P<0.05), as shown in Table 4.

[0086] Table 4 Comparison of serum oxidative stress levels among the three groups before and after treatment

[0087]

[0088]

[0089] Note: Compared with the control group after treatment, * P<0.05; compared with the Huoxue Rongluo Pills group after treatment, ▲ P<0.05; compared with the group before treatment, # P<0.05.

[0090] 6. Conclusion

[0091] In this study, Huoxuerongluo Pills and Huoxuerongluo Composition Tablets (i.e., Composition Composition Tablets) were added to conventional western medicine treatment, and it was found that Huoxuerongluo Pills and Huoxuerongluo Tablets could significantly reduce the symptoms of neurological deficits in patients with acute cerebral infarction and improve the quality of life of patients with cerebral infarction. Serum oxidative stress levels in patients with cerebral infarction were measured. GSH-Px is an important antioxidant enzyme in the body, and SOD is an antioxidant enzyme that can inhibit lipid peroxidation. Its concentration can reflect the body's antioxidant capacity. Increased MDA levels usually indicate oxidative stress or free radical damage in the body. The results of this study showed that after treatment, the MDA levels of all patients were significantly reduced, and the levels of GSH-Px and SOD were significantly increased. The MDA levels were lower and the GSH-Px and SOD levels were higher when Huoxuerongluo Pills and Huoxuerongluo Composition Tablets were added. Therefore, the serum oxidative stress level and neurological function of patients with cerebral infarction treated with Huoxue Rongluo Pills and Huoxue Rongluo component tablets combined with basic Western medicine treatment were significantly improved, which can further enhance their daily living ability, thereby improving the overall treatment efficiency of patients and the Huoxue Rongluo component tablets (i.e., component combination tablets) have better efficacy.

[0092] Example 2

[0093] 1. Main drugs and reagents

[0094] Huoxuerongluo Pills: The active ingredients are 30mg of honeysuckle vine, 30mg of Photinia serrulata, 10mg of peach kernel, 10mg of Vaccaria segetalis, 15mg of Gastrodia elata, 15mg of Scrophularia ningpoensis, 10mg of Paeonia lactiflora, 15mg of Papaya, 10mg of Acorus calamus, 10mg of Angelica dahurica, 6mg of Gleditsia sinensis, 10mg of Gentiana macrophylla, and 0.3mg of Borneol. Provided by the Department of Pharmaceutical Preparation of the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine, the conventional dosage for patients with cerebral infarction is 15g / d. According to the body surface area-dose conversion method, the pills were dissolved in distilled water to prepare a liquid of about 1.35g / kg.

[0095] Huoxuerongluo component tablets: that is, the component composition, composed of 2mg of isoimperatorin, 4mg of chlorogenic acid, 20mg of barleyson glycoside A, 2mg of cinnamic acid, 8mg of paeoniflorin, 5mg of paeoniflorin, 4mg of loganin and 8mg of β-asarone, totaling 51mg, in the form of cyclodextrin inclusion compound. After active ingredient pretreatment, dry mixing, wet granulation, drying and tableting, Huoxuerongluo tablets with a total tablet weight of 101mg are prepared. The conventional dosage for patients with cerebral infarction is 9 tablets / d (909mg / d). According to the body surface area-dose conversion method, the tablets are dissolved in distilled water to prepare a drug solution of about 81.8mg / kg.

[0096] 2. Animal Grouping and Dosing

[0097] SPF Sprague Dawley rats, weighing (300±10) g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., license number SCXK: 2019-0004. Rats were kept in the animal room of the First Affiliated Hospital of Hunan University of Chinese Medicine, with a temperature of (25±1)℃ and a relative humidity of (50±10)%, a day and night cycle of 12h, free access to food and water, and adaptive feeding for 1 week before the experiment. All operations in this experiment were carried out in strict accordance with animal ethics requirements and approved by the Experimental Animal Ethics Committee of the First Affiliated Hospital of Hunan University of Chinese Medicine (approval number ZYFY20231101-11).

[0098] After 1 week of adaptive feeding, SD rats were randomly divided into sham group (Sham group), model group (Model group), Huoxuerongluo group piece group (ZFPW group) and Huoxuerongluo pill group (HXRLW group) according to body weight, with 10 rats in each group. MCAO / R injury model of rats was established in model group and each drug group: rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), placed on a heating pad, and the rectal temperature was maintained at 37°C. The skin of the front neck was prepared, and an incision was made in the anterior midline to expose the common carotid artery, internal carotid artery and external carotid artery. The distal end of the external carotid artery was ligated, and the distal end of the internal carotid artery and the proximal end of the common carotid artery were clamped. The thread plug was sent into the middle cerebral artery through the internal carotid artery, and the thread plug was pushed 18-20 mm. After 2 hours of ischemia, the thread plug was pulled out and the port was ligated to achieve reperfusion. In the control group, the blood vessels were only isolated without thread plugs. Each drug group was gavaged with the corresponding drug, and the Sham group and the Model group were gavaged with an equal volume of normal saline, once a day for 7 consecutive days. On the 8th day, the rats were tested and samples were collected.

[0099] 3. Animal Experiment Methods and Results

[0100] 3.1 Modified neurological severity score (mNSS)

[0101] After ischemia-reperfusion in rats, mNSS was measured to evaluate the function of the animal's nervous system: normal rats were scored 0 points, mNSS of 1-6 points indicated mild damage, 7-12 points indicated moderate damage, and 13-18 points indicated severe damage. The mNSS of rats was measured on the 1st, 3rd, and 7th days of administration. Scoring criteria: 1 point for flexion of the forelimbs and hindlimbs; 1 point for inability to walk in a straight line, 2 points for turning in circles to the paretic side, and 3 points for falling to the paretic side; 1 point for loss of auricular reflex, corneal reflex, startle reflex, and dystonia; 1 point for placement test and proprioception test; 1 point for grasping the balance beam, 2 points for falling from the balance beam with one limb, 3 points for falling from the balance beam with two limbs, 4 points for staying on the balance beam for more than 40 seconds and finally falling, 5 points for staying on the balance beam for 20 seconds but finally falling; the highest score was 18 points.

[0102] Neurological impairment was evaluated by measuring the mNSS score 7 days after the successful establishment of the MCAO / R model. The mNSS score results showed that the rats in the Model group had more severe neurological dysfunction, and the mNSS scores of the ZFPW group and the HXRLW group were reduced (P < 0.01 or P < 0.05), among which the ZFPW group was more significantly reduced than the HXRLW group (P < 0.05), indicating that Huoxue Rongluo Tablets better improved the neurological deficit symptoms of MCAO / R rats. (As shown in Table 5)

[0103] Table 5 mNSS scores of rats in each group (mean±SD, n=10)

[0104]

[0105] Note: Compared with the Model group, **P<0.01; *P<0.05; compared with the HXRLW group, #p<0.05.

[0106] 3.2 TTC staining

[0107] Rats were anesthetized, brain tissue slices were taken, frozen at -80℃ for 6-7min, and the brain was cut into 5 slices every 2mm coronally. The prepared 2% TTC solution was preheated in a 37℃ incubator, and the cut brain slices were placed in the dye solution for staining for 15min. After the brain slices were evenly stained, they were taken out and fixed in 4% paraformaldehyde for 24h. The fixed brain slices were placed on filter paper and photographed with a digital camera, and the Infact Volume percentage was analyzed using Image J 1.53a software. Infact Volume (%) = (cerebral infarction area × thickness) / (whole brain area × thickness).

[0108] The infarct area of ​​rats after MCAO / R modeling increased significantly, while the infarct volume of ZFPW group and HXRLW group after MCAO / R was reduced compared with that of Model group (P<0.01 or P<0.05), and the reduction in ZFPW group was more significant than that in HXRLW group (P<0.05), indicating that Huoxue Rongluo tablets are more effective in reducing the volume of cerebral infarction in MCAO / R rat model. Figure 1 and Table 6)

[0109] Table 6 Statistical results of TTC staining infarct area of ​​brain tissue of rats in each group (mean ± SD, n = 5)

[0110]

[0111] Note: Compared with the Model group, **P<0.01; *P<0.05; compared with the HXRLW group, #p>0.05.

[0112] 3.3HE staining

[0113] Take 4% paraformaldehyde to fix rat brain tissue, embed it in paraffin, and make coronal sections with a thickness of 4μm, and fix it on a slide. Put the sections in xylene, and change the xylene repeatedly until the sections are completely transparent. Put the sections in 100%, 95%, 80%, and 70% alcohol in turn, and change each alcohol once, each time for 3-5 minutes. Perform hematoxylin-eosin staining or Nissl staining for 5-10 minutes. Put the sections in 70%, 80%, 95%, and 100% alcohol in turn, and change each alcohol once, each time for 3-5 minutes. Finally, use xylene to make it transparent and neutral resin to seal the sections, and take pictures on the Pannoramic MIDI pathology scanner (3D HISTECN). Image J 1.53a software was used to analyze the pathological changes of brain tissue, and the inflammatory cell infiltration score was used to evaluate the HE staining: 0 points indicated no inflammatory cell infiltration or only a very small amount of inflammatory cells; 1 point indicated mild inflammatory cell infiltration, with sparse distribution of inflammatory cells, mainly concentrated around blood vessels; 2 points indicated moderate inflammatory cell infiltration, with dense and widespread distribution of inflammatory cells, not limited to around blood vessels; 3 points indicated severe inflammatory cell infiltration, with very dense and widespread distribution of inflammatory cells, which may form nodules or aggregates; 4 points indicated extremely severe inflammatory cell infiltration, with almost the entire field of view occupied by inflammatory cells, which may be accompanied by obvious tissue damage.

[0114] HE staining showed that the cerebral cortex of rats in the Model group showed severe pathological damage, necrosis and inflammatory cell infiltration, and the neurons were damaged, while the pathological damage of brain tissues of rats in the ZFPW and HXRLW groups was significantly improved compared with that in the Model group (P < 0.01 or P < 0.05), and the ZFPW group showed a trend of alleviation compared with the HXRLW group. Figure 2 and Table 7)

[0115] Table 7 Inflammatory cell infiltration in rat brain tissue stained with HE

[0116]

[0117] Note: Compared with the Model group, **P<0.01; *P<0.05; compared with the HXRLW group, #p>0.05.

[0118] 3.4. Ferrous ion determination

[0119] The homogenate of brain tissue of each rat was obtained and operated according to the instructions of the kit. The absorbance (A) value was measured using a multifunctional microplate reader (SpectraMax iD5) and the ferrous ion (593 nm) level was calculated.

[0120] Fe in brain tissue of rats in Model group 2+ The level of Fe was significantly increased in the ZFPW group and the HXRLW group, while the level of Fe was significantly increased in the ZFPW group and the HXRLW group after MCAO / R.2+ The level of leukemia increased (P<0.01), and the inhibitory effect of ZFPW group was stronger (P<0.05). (As shown in Table 8)

[0121] Table 8 Fe in brain tissue of rats in each group 2+ Content (mean±SD, n=5)

[0122]

[0123] Note: Compared with the Model group, **P<0.01; *P<0.05; compared with the HXRLW group, #p>0.05.

[0124] Transmission electron microscopy

[0125] The rat brain tissue was obtained and rinsed with PBS, fixed at 4°C (2.5% glutaraldehyde, 12h), rinsed with PBS, dehydrated, fixed (1% osmium acid, 2h), rinsed with PBS, dehydrated with acetone (30%, 50%, 70%, 80%, 95%, 100%), infiltrated and embedded (Epon812), ultrathinly sectioned (50-70nm), and stained (uranyl acetate and lead citrate). Then, images were collected and analyzed under a transmission electron microscope.

[0126] In the images of the Sham group, the mitochondrial morphology should remain intact. The mitochondrial double membrane is clear, and the internal cristae structure is tight and orderly arranged. There is no obvious mitochondrial swelling, cristae breakage or disappearance, which belongs to the normal mitochondrial morphology without damage. In the Model group, the mitochondria showed obvious signs of damage. It may be observed that the mitochondria are swollen, the cristae structure is blurred or broken, the double membrane is not clear, and vacuolation occurs inside the mitochondria. The mitochondria in the HXRLW group and the ZFPW group showed certain signs of recovery compared with the Model group. The mitochondrial swelling was relatively mild, and some cristae structures were still visible, but there was still a certain membrane rupture phenomenon; see for details. Figure 3 .

[0127] Immunofluorescence staining

[0128] The rat brain tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into coronal sections with a thickness of 4 μm. After dewaxing and rehydration with xylene and different concentrations of gradient alcohol, antigen retrieval was performed in antigen retrieval solution, blocked with 3% BSA, and GPX4 (1:100) and ACSL4 (1:100) were diluted and added to the sections, and incubated at 4°C overnight. The next day, the sections were incubated with fluorescently labeled secondary antibodies (1:200) for 1 to 2 hours, observed and photographed under an inverted fluorescence microscope (Olympus IX73) to detect the localization and quantitative expression of GPX4 and ACSL4 in rat brain tissue. Image J1.53a software was used to analyze the average fluorescence intensity of GPX4 and ACSL4.

[0129] The results of immunofluorescence double staining showed that the expression of GPX4 in the hippocampus of the Model group was significantly decreased, the expression of ACSL4 was significantly increased, and the average fluorescence intensity of GPX4 / ACSL4 showed a significant decrease. However, the average fluorescence intensity of GPX4 / ACSL4 after MCAO / R was significantly increased in both the HXRLW group and the ZFPW group (P < 0.01 or P < 0.05), and the upregulation trend in the ZFPW group was more obvious than that in the HXRLW group (P < 0.01 or P < 0.05). (As shown in Figure 2, the expression of GPX4 in the hippocampus of the Model group was significantly decreased, the expression of ACSL4 was significantly increased, and the average fluorescence intensity of GPX4 / ACSL4 was significantly decreased in the HXRLW group and the ZFPW group after MCAO / R (P < 0.01 or P < 0.05). Figure 4 and Table 9)

[0130] Table 9 Average fluorescence intensity of GPX4 / ACSL4 in brain tissue of rats in each group (mean±SD, n=5)

[0131]

[0132] Note: Compared with the Model group, **P<0.01; *P<0.05; compared with the HXRLW group, #p>0.05.

[0133] 3.7.Western Blot

[0134] After the brain tissue was grouped and processed, total protein was extracted from the rat brain tissue, and the extracted protein was quantified using a BCA protein quantification kit. The quantified protein samples were denatured, and protein separation was performed using SDS-PAGE gel. The denatured protein was transferred from the SDS-PAGE gel to PVDF, and the transferred PVDF membrane was placed in a blocking solution to block nonspecific binding sites. The primary antibodies of GPX4 (1:3000), ACSL4 (1:2000), and β-actin (1:4000) were diluted and added to the PVDF membrane and incubated overnight at 4°C. After washing 3 times, the corresponding secondary antibody (1:8000) was diluted and incubated with the PVDF membrane at room temperature for 2 hours. After washing with TBST for 3 times, ECL luminescence reagent was added and the protein expression level of the target band was analyzed using a gel imaging system (AmershamImager 600). Image J 1.53a software was used to detect the gray value of the image and analyze it. The relative expression of the protein was expressed as the ratio of the gray value of the target protein to that of β-actin.

[0135] Western Blot results showed that after MCAO / R (Model group), the GPX4 protein level was significantly decreased, and the ACSL4 protein level was significantly increased, indicating that GPX4 was inhibited and ACSL4 was activated; the compound preparation Huoxue Rongluo Pill II extract (HXRLW group) and Huoxue Rongluo Pill II component solution (ZFPW group) could promote the expression of GPX4 protein level after MCAO / R, and inhibit the expression of ACSL4, thereby inhibiting the progression of ferroptosis and reducing ferroptosis damage after MCAO / R in rats (such as Figure 5 and Table 10).

[0136] Table 10 Relative protein expression of GPX4 and ACSL4 in brain tissue of rats in each group (mean ± SD, n = 5)

[0137]

[0138] Note: Compared with the Model group, **P<0.01; *P<0.05; compared with the HXRLW group, #p>0.05.

[0139] 4. Conclusion

[0140] In our study, we found that the solution prepared by Huoxuerongluo Pills and Huoxuerongluo Tablets significantly reduced the mNSS score of MCAO / R rats at 7 days, reduced the infarct volume, alleviated pathological damage, increased the number of neurons, and improved the symptoms of neurological deficit after ischemic stroke. 2+ The iron accumulation level induced by MCAO / R injury in rats was evaluated after intervention with the two drugs. In addition, immunofluorescence double staining and Western blot were used to analyze the expression of ferroptosis-related proteins in the GPX4 / ACSL4 pathway. The results showed that Huoxue Rongluo Pills and Huoxue Rongluo Tablets could reduce the Fe content in the brain tissue of MCAO / R rats. 2+ level, and can activate GPX4, inhibit ACSL4, and thus reduce ferroptosis after MCAO / R in rats. At the same time, we used transmission electron microscopy to observe the ultrastructure in rat brain tissue and found that after MCAO / R, the mitochondrial structure was damaged, the volume was reduced, the double membrane density was increased, the mitochondrial cristae were reduced, and the mitochondrial outer membrane was ruptured, and the Huoxuerongluo Pills and Huoxuerongluo Tablets liquids could partially reverse the mitochondrial structural damage. Therefore, with respect to the effects of Huoxuerongluo Pills and Huoxuerongluo Tablets liquids in ischemic stroke, it was found that they could reduce iron accumulation, inhibit ferroptosis by regulating the GPX4 / ACSL4 / pathway, and improve mitochondrial structural and functional damage. And Huoxuerongluo group tablets (the composition combination of the present invention) have better efficacy.

[0141] The combination of Chinese medicine components is an optimization strategy based on the combination of traditional Chinese medicine theory and modern pharmacology. Compared with traditional Chinese medicine compound preparations, the combination of components can achieve more accurate and efficient therapeutic effects by extracting, purifying and recombining the effective ingredients in Chinese medicine, providing new mechanistic evidence for the combination of Huoxue Tongluo Pills in the treatment of ischemic stroke.

[0142] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A pharmaceutical composition for treating ischemic stroke, characterized in that: Its active ingredients include chlorogenic acid, loganin, peonidin, paeoniflorin, barisenoside A, cinnamic acid, β-asarone and isoimperatorin.

2. The pharmaceutical composition for treating ischemic stroke according to claim 1, characterized in that: By weight, the invention comprises 1-5 parts of chlorogenic acid, 2-8 parts of loganin, 3-10 parts of paeoniflorin, 5-15 parts of paeoniflorin, 10-30 parts of barisenoside A, 1-5 parts of cinnamic acid, 5-15 parts of β-asarone and 1-5 parts of isoimperatorin.

3. The pharmaceutical composition for treating ischemic stroke according to claim 2, characterized in that: Calculated by weight, the invention comprises 2 parts of chlorogenic acid, 4 parts of loganin, 5 parts of paeoniflorin, 8 parts of paeoniflorin, 20 parts of parisonoside A, 2 parts of cinnamic acid, 8 parts of β-asarone and 2 parts of isoimperatorin.

4. The pharmaceutical composition for treating ischemic stroke according to any one of claims 1 to 3, characterized in that: Pharmaceutically acceptable excipients are also included.

5. The pharmaceutical composition for treating ischemic stroke according to claim 4, characterized in that: The dosage forms of the pharmaceutical composition include powder, soft capsule, granule, pill, tablet, decoction and hard capsule.

6. The pharmaceutical composition for treating ischemic stroke according to claim 4, characterized in that: The dosage of the auxiliary material is 1:0.95-1.05 based on the total mass of the active ingredient.

7. The pharmaceutical composition for treating ischemic stroke according to claim 4, characterized in that: The auxiliary materials include at least one of fillers, adhesives, lubricants and solubilizers.

8. The pharmaceutical composition for treating ischemic stroke according to claim 7, characterized in that: The auxiliary materials include at least one of starch, dextrin, sucrose, milk powder, sweetener, mannitol, lactose, cyclodextrin, disodium EDTA, magnesium stearate, calcium stearate, talcum powder, flavor and sodium lauryl sulfate.

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