Application of artemisia apiacea extract in prevention and / or treatment of cerebral arterial thrombosis

Abstract

The invention provides application of an artemisia apiacea extract in preparation of a medicine for preventing and / or treating cerebral arterial thrombosis. The artemisia apiacea extract is a methanol extract of artemisia apiacea. The artemisia apiacea extract inhibits neuronal ferroptosis by regulating glutamic acid metabolism so as to relieve nerve cell injury of rats suffering from cerebral arterial thrombosis, so that the effect of preventing and / or treating cerebral arterial thrombosis is achieved.

Description

Technical Field

[0001] This application relates to the application of artemisia annua extract in the prevention and / or treatment of ischemic stroke, and belongs to the field of traditional Chinese medicine technology. Background Technology

[0002] Ischemic stroke (IS) is a disease characterized by necrosis or softening of localized brain tissue due to ischemia and hypoxia, resulting in corresponding neurological symptoms such as ataxia, slurred speech, unilateral gaze palsy, blurred vision, dizziness, headache, vomiting, altered consciousness, or seizures. Epidemiological surveys show that stroke has become the second leading cause of death and disability worldwide, with ischemic stroke patients accounting for 60%-80% of these cases. Ischemic stroke poses a significant threat to public health due to its high incidence, high mortality, high disability rate, high recurrence rate, and high economic burden. Therefore, finding safe and effective traditional Chinese medicine treatments is of great significance for the prevention and treatment of IS. Recent studies have shown that ferroptosis is closely related to neuronal cell damage in ischemic stroke, and inhibiting ferroptosis can counteract the excitotoxicity of glutamate-induced neurons. During IS, glutamate accumulation plays a crucial role in the process of neuronal ferroptosis. Within minutes of ischemic stroke onset, a large amount of glutamate is released, leading to excessive accumulation of glutamate in brain regions such as the cerebral cortex and hippocampus. Excessive glutamate reduces the uptake of cystine by the cysteine / glutamate exchange transporter (system Xc–). Cysteine, a byproduct of cysteine ​​breakdown, is an important amino acid for the synthesis of glutathione (GSH). The inability to sustain GSH synthesis results in intracellular GSH depletion, increased reactive oxygen species (ROS), and increased lipid peroxidation, ultimately leading to ferroptosis. Therefore, regulating ferroptosis induced by excessive glutamate accumulation may be an important pathway to combat neuronal damage in ischemic stroke.

[0003] Artemisia annua L., a plant belonging to the genus Artemisia in the family Asteraceae, is a traditional Chinese medicine. It contains various active ingredients, including sesquiterpenes, volatile oils, flavonoids, fatty acids, polyphenols, coumarins, and esters. Sesquiterpenes are the main and most effective components of Artemisia annua. Artemisia annua extract and its main component, sesquiterpenes, have a wide range of pharmacological effects, including anti-inflammatory, antibacterial, antiparasitic, antiviral, antitumor, and hypoglycemic effects. However, there are no reports on the application of Artemisia annua extract in the prevention and / or treatment of ischemic stroke. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides the application of artemisia annua extract in the prevention and / or treatment of ischemic stroke.

[0005] As a first aspect of the invention, the invention provides the use of artemisia annua extract in the preparation of medicaments for the prevention and / or treatment of ischemic stroke.

[0006] According to any application of the first aspect of the present invention, the artemisinin extract is used in regulating glutamate metabolism.

[0007] According to any application of the first aspect of the present invention, the artemisinin extract is used in inhibiting neuronal ferroptosis.

[0008] According to any application of the first aspect of the present invention, the artemisia extract is an organic solvent extract of artemisia.

[0009] Preferably, the organic solvent is selected from any one or more of methanol, ethanol, n-butanol, acetone, ethyl acetate, chloroform, and petroleum ether.

[0010] More preferably, the organic solvent is methanol.

[0011] According to any application of the first aspect of the present invention, the artemisia extract is prepared by conventional extraction methods, including maceration extraction, ultrasonic extraction, percolation extraction, microwave extraction, and supercritical CO2 extraction.

[0012] As a second aspect of the invention, the invention provides the use of artemisinin in the preparation of medicaments for the prevention and / or treatment of ischemic stroke.

[0013] As a third aspect of the invention, the invention provides a composition for the prevention and / or treatment of ischemic stroke, the composition comprising an effective amount of artemisinin extract and / or artemisinin and a pharmaceutically acceptable carrier.

[0014] According to any one of the third aspects of the present invention, the artemisia extract is an organic solvent extract of artemisia.

[0015] Preferably, the organic solvent is selected from any one or more of methanol, ethanol, n-butanol, acetone, ethyl acetate, chloroform, and petroleum ether.

[0016] More preferably, the organic solvent is methanol.

[0017] According to any one of the third aspects of the present invention, the artemisia extract is prepared by conventional extraction methods, including maceration extraction, ultrasonic extraction, percolation extraction, microwave extraction, and supercritical CO2 extraction.

[0018] According to any one of the third aspects of the present invention, the composition is prepared into, but is not limited to, injections, capsules, tablets, granules, gels, sustained-release formulations, oral liquids, pellets, or nano-formulations. The pharmaceutically acceptable carrier includes: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrices. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silica, etc.; suspending agents include: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch paste, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.

[0019] As a fourth aspect of the present invention, the present invention provides a method for preventing and / or treating ischemic stroke, the method comprising administering an effective amount of artemisinin extract to the subject.

[0020] According to any of the fourth aspects of the present invention, the artemisia extract is an organic solvent extract of artemisia.

[0021] Preferably, the organic solvent is selected from any one or more of methanol, ethanol, n-butanol, acetone, ethyl acetate, chloroform, and petroleum ether.

[0022] More preferably, the organic solvent is methanol.

[0023] According to any of the methods of the fourth aspect of the present invention, the artemisia extract is prepared by conventional extraction methods, including maceration extraction, ultrasonic extraction, percolation extraction, microwave extraction, and supercritical CO2 extraction.

[0024] According to any of the fourth aspects of the present invention, the effective amount of artemisia annua extract is 12.5 mg to 100 mg per kilogram of subject body weight per day; preferably, the effective amount of artemisia annua extract is 25 mg to 100 mg per kilogram of subject body weight per day; preferably, the effective amount of artemisia annua extract is 50 mg per kilogram of subject body weight per day.

[0025] The "artemisia" mentioned in this invention is listed in Part I of the 2020 edition of the Chinese Pharmacopoeia, and is the dried aerial part of Artemisia annua L., a plant of the Asteraceae family.

[0026] The beneficial effects of this invention: This study shows that 24 hours after ischemic stroke in rats, the rats' neurological deficit scores significantly increased, exhibiting symptoms such as hemiplegia, unidirectional spinning, and flexion of the forelimbs to the opposite side when the tail is lifted. TTC staining revealed large areas of pale ischemic areas in the brain tissue. HE staining and Nissl body staining revealed a decrease in the number of neurons, loose arrangement, abnormal morphology, vacuolation at cell edges, and blurred Nissl bodies. Rats pre-treated with Artemisia annua extract showed reduced neurological deficit scores, alleviated various neurological symptoms, and significantly smaller ischemic areas in the brain tissue compared to the model group. The degree of microstructural damage was reduced, the number of cells was greater, the morphology was intact, the arrangement was more compact, and the Nissl bodies were clearer and more obvious. These results indicate that Artemisia annua extract has a protective effect on the morphology and function of rat brain tissue after ischemic stroke, and has positive significance for the prevention and progression of ischemic stroke. In addition, this study also found that the effect of Artemisia annua extract on ischemic stroke is significantly better than that of artemisinin monomer, which also reflects the advantages of the synergistic effect of multiple components of traditional Chinese medicine extract. Attached Figure Description

[0027] Figure 1 The neurological deficit scores of rats in each group were calculated (n=6); compared with the sham-operated group, ** P<0.01, compared with the model group, # P<0.05, ## P<0.01.

[0028] Figure 2 TTC staining images of rat brain tissue from each group (n=6).

[0029] Figure 3 The infarct volume of rats in each group (n=6); compared with the sham-operated group, ** P<0.01, compared with the model group, # P<0.05, ## P<0.01.

[0030] Figure 4 The pathological changes of hippocampal tissue in the ischemic area of ​​rats in each group (HE staining, ×200) are shown; A. sham-operated group; B. model group; C. Ginkgo biloba extract group; D. Artemisia annua extract 12.5 mg / kg; E. Artemisia annua extract 25 mg / kg; F. Artemisia annua extract 50 mg / kg; G. Artemisia annua extract 100 mg / kg.

[0031] Figure 5Nissl staining (×200) was performed on the hippocampal tissue of the ischemic area of ​​rats in each group; A. sham-operated group; B. model group; C. Ginkgo biloba extract group; D. Artemisia annua extract 12.5 mg / kg; E. Artemisia annua extract 25 mg / kg; F. Artemisia annua extract 50 mg / kg; G. Artemisia annua extract 100 mg / kg.

[0032] Figure 6 Fe in the brain tissue of rats in each group 2+ The content of MDA, GSH, and SOD (n=6); among which, A, B, C, and D are Fe, MDA, GSH, and SOD, respectively. 2+ Content, MDA content, GSH content, SOD content; compared with the sham surgery group, ** P<0.01, compared with the model group, # P<0.05, ## P<0.01.

[0033] Figure 7 The expression levels of SLC7A11, GPX4, EAAT1, EAAT2, GS, and PAG mRNA in the brain tissue of rats in each group were measured (n=6); where A, B, C, D, E, and F represent the expression levels of SLC7A11, GPX4, EAAT1, EAAT2, GS, and PAG mRNA, respectively; compared with the sham-operated group, * P<0.05, ** P<0.01, compared with the model group, # P<0.05, ## P<0.01.

[0034] Figure 8 Electrophoresis images of SLC7A11, GPX4, and FTH1 in the brain tissue of rats in each group; where 1. sham-operated group; 2. model group; 3. Ginkgo biloba extract group; 4. Artemisia annua extract 12.5 mg / kg group; 5. Artemisia annua extract 25 mg / kg group; 6. Artemisia annua extract 50 mg / kg; 7. Artemisia annua extract 100 mg / kg.

[0035] Figure 9 The expression levels of SLC7A11, GPX4, and FTH1 proteins in the brain tissue of rats in each group (n=3); B, C, and D represent the expression levels of SLC7A11, GPX4, and FTH1 proteins, respectively; compared with the sham-operated group, * P<0.05, ** P<0.01, compared with the model group, # P<0.05, ## P<0.01.

[0036] Figure 10 TTC staining images of rat brain tissue from each group (n=8).

[0037] Figure 11 The effect of Artemisia annua extract on glutamate-induced HT22 cell survival (n=6); compared with the normal control group, * P<0.05, ** P<0.01, compared with the model group, # P<0.05, ## P<0.01.

[0038] Figure 12 The levels of MDA, GSH, and SOD in HT22 cells of each group after glutamate treatment (n=6) were compared with those of the normal control group. * P<0.05, ** P<0.01, compared with the model group, # P<0.05, ## P<0.01.

[0039] Figure 13 The expression levels of SLC7A11 and GPX4 mRNA in HT22 cells of each group after glutamate treatment (n=6); compared with the normal control group, * P<0.05, compared with the model group, # P<0.05, ## P<0.01.

[0040] Figure 14 The expression levels of HT22 cell-related proteins in each group after glutamate treatment (n=3); compared with the normal control group, * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01. Detailed Implementation

[0041] The present invention is further described below through specific embodiments, but the present invention is not limited to the following embodiments. Modifications, combinations, or substitutions made to the present invention within the scope of the present invention or without departing from the content, spirit, and scope of the present invention will be obvious to those skilled in the art and are included within the scope of the present invention.

[0042] Example 1: Preparation of Artemisia annua extract

[0043] Artemisia annua was extracted twice with 20 times the amount of methanol using ultrasonic extraction, each time for 15 minutes. The extract was then concentrated using a rotary evaporator to obtain Artemisia annua extract.

[0044] The extract yield is 18.86g per kg of Artemisia annua technical grade. Store at 4℃ for later use.

[0045] Example 2: Preparation of Artemisia annua extract

[0046] Artemisia annua was extracted twice with 10 times the amount of ethanol using ultrasonic extraction, each time for 15 minutes; the extract was then concentrated using a rotary evaporator to obtain Artemisia annua extract.

[0047] Example 3: Preparation of Artemisia annua extract

[0048] Artemisia annua was extracted twice with 30 times the amount of n-butanol using ultrasonic extraction, each time for 15 minutes; the extract was then concentrated using a rotary evaporator to obtain Artemisia annua extract.

[0049] Example 4: Preparation of Artemisia annua extract

[0050] Artemisia annua was extracted twice with 20 times the amount of acetone using ultrasonic extraction, each time for 15 minutes; the extract was then concentrated using a rotary evaporator to obtain Artemisia annua extract.

[0051] Example 5: Preparation of Artemisia annua extract

[0052] Artemisia annua was extracted twice with 10 times the amount of ethyl acetate using ultrasonic extraction, each time for 15 minutes; the extract was then concentrated using a rotary evaporator to obtain Artemisia annua extract.

[0053] Example 6: Preparation of Artemisia annua extract

[0054] Artemisia annua was extracted twice with chloroform at a concentration of 30 times, each time for 15 minutes. The extract was then concentrated using a rotary evaporator to obtain Artemisia annua extract.

[0055] Example 7 Preparation of Artemisia annua extract

[0056] Artemisia annua was extracted twice with 20 times the amount of petroleum ether using ultrasonic extraction, each time for 15 minutes; the extract was then concentrated using a rotary evaporator to obtain Artemisia annua extract.

[0057] Example 8: Preparation of Artemisia annua extract tablets

[0058] Take any of the artemisia annua extracts prepared in Examples 1-7, add tablet excipients, and prepare artemisia annua extract tablets according to conventional tablet preparation processes.

[0059] Example 9: Preparation of Artemisia annua extract granules

[0060] Artemisia annua extract prepared in any of Examples 1-7 was added to granule excipients and prepared into Artemisia annua extract granules according to conventional granule preparation process.

[0061] Example 10: Preparation of Artemisia annua extract capsules

[0062] Take any of the Artemisia annua extracts prepared in Examples 1-7, add granulation excipients, granulate according to conventional processes, and fill into capsules to prepare Artemisia annua extract capsules.

[0063] Preparation of Artemisia annua Extract Granules in Example 11

[0064] Take the Artemisia annua extract prepared in any one of Examples 1-7, add excipients for injection, and prepare an Artemisia annua extract injection according to the conventional preparation process of injections.

[0065] Effect Experiment (I) of Artemisia annua Extract in Preventing and / or Treating Ischemic Stroke in Example 12

[0066] 1 Materials and Methods

[0067] 1.1 Animals

[0068] 105 SPF-grade healthy male SD rats, 8-10 weeks old, weighing 280±30 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animal production license number is: SYXK (Beijing) 2022-0052. The animals were housed in the Animal Experiment Center of Beijing University of Chinese Medicine, with a 12-hour day-night cycle, room temperature (20±2)°C, and relative humidity of 50% - 60%. They were allowed to eat and drink freely. They were adaptively fed for 3 days.

[0069] 1.2 Drugs

[0070] The Artemisia annua Chinese herbal pieces used in the experiment were purchased from Beijing Tongrentang Changyang Pharmacy Co., Ltd., batch number: 20210827, and were identified by teachers from the Chinese Medicine Teaching and Research Section of the School of Chinese Medicine, Beijing University of Chinese Medicine. They were prepared into Artemisia annua extract according to the method in Example 1 and stored in a 4°C refrigerator for later use.

[0071] 1.3 Main Reagents

[0072] Middle cerebral artery ischemia-thrombosis line (Beijing Xinong Technology Co., Ltd., catalog number: 2634A4); Triphenyltetrazolium chloride (TTC, Beijing Solarbio Technology Co., Ltd., catalog number: G3005); Ginkgo biloba extract tablets (Dr. Willmar Schwabe GmbH & Co. KG, Germany, catalog number: G832683); GSH, superoxide dismutase (SOD), and tissue iron assay kits (Nanjing Jiancheng Bioengineering Institute, catalog numbers A006-2-1, A001-3-2, and A039-2-1 respectively); Malondialdehyde (MDA) test kit (KGI Biotechnology, catalog number: KGA7101-100); Total RNA small-volume extraction kit (Guangzhou Meiji Biotechnology Co., Ltd., catalog number: R4111-02); Reverse transcription kit (Thermo Fisher Scientific, USA). Scientific (K1622); Real-time quantitative polymerase chain reaction kit (Suzhou Nearshore Protein Technology Co., Ltd., E096-01A); BCA (bicinchoninic acid) protein concentration assay kit (Shanghai Beyotime Biotechnology Co., Ltd., P0009); Protease and phosphatase inhibitor mixture (Synthetic Biotechnology Co., Ltd., P002); Recombinant solute carrier family 7 member 11 (SLC7A11, xCT) antibody (Abcam, 175186); Glutathione peroxidase 4 (GPX4) antibody (Proteintech, 30388-1-AP); Ferritin heavy chain 1 (FTH1) antibody (CST Biotech, 3998S); GAPDH Rabbit PolyAb (Proteintech, USA, 10494-1-ap); Goat anti-rabbit IgG H&L (Proteintech, USA, Catalog No.: SA00001-2); Ultrasensitive ECL chemiluminescence reagent kit (Synsemi Biotechnology Co., Ltd., P10100).

[0073] 1.4 Grouping and Dosing

[0074] One hundred and five male SD rats were randomly divided into seven groups using a random number table: sham-operated group, model group, Ginkgo biloba extract group, Artemisia annua extract group (12.5 mg / kg), Artemisia annua extract group (25 mg / kg), Artemisia annua extract group (50 mg / kg), and Artemisia annua extract group (100 mg / kg), with 15 rats in each group. The Ginkgo biloba extract group received the drug at a dose of 50 mg / kg, while the Artemisia annua extract groups received the drugs at doses of 12.5 mg / kg, 25 mg / kg, 50 mg / kg, and 100 mg / kg, respectively. All drug administration groups received a uniform mixture of the drug and 0.5% sodium carboxymethyl cellulose (CMC-Na) suspension via gavage for six days. The sham-operated group and model group received CMC-Na suspension via gavage. Modeling was initiated 30 minutes after drug administration on day 6.

[0075] 1.5 Modeling Method

[0076] Preoperative fasting (but water intake is permitted) for 12 hours was used to establish a rat model of middle cerebral artery occlusion (MCAO) using the suture occlusion method. An anesthetic was prepared using a 1:1 mixture of 10% chloral hydrate and 20% urethane (Zhang Quanpeng, Wang Hui, Chen Dan, et al. Comparison and application of the effects of chloral hydrate, urethane, and their 1:1 mixture in anesthesia of SD rats [J]. Modern Biomedical Progress, 2011, 11(07): 1208-1212), administered via intraperitoneal injection at a dose of 0.5 mL / 100 g. The deeply anesthetized rats were incised 1-2 cm along the midline of the neck to expose the left common carotid artery. This artery was bluntly separated from the left fascia, and the internal and external carotid arteries were isolated. The external carotid artery was briefly clamped with an arterial clamp. After ligating the common carotid artery proximal to the heart, a V-shaped hole was cut and a suture occluded was inserted. The suture was slightly adjusted to enter the internal carotid artery, stopping when the depth reached approximately 19 mm. The distal end of the small incision was then ligated, excess sutures were cut, the wound was disinfected with antibiotics, and sutured. The rats were placed on a warming pad to keep them warm post-operatively until the anesthesia wore off. The sham surgery group underwent the same procedures as the rats in the sham surgery group, except that no sutures were inserted.

[0077] 1.6 Zea Longa scoring method for detecting neurological function in rats

[0078] Neurological deficit assessment was performed 24 hours after modeling. The Zea-Longa neurological deficit assessment scale is a 5-point scale from 0 to 4: 0 points, no neurological damage; 1 point, adduction and flexion of the contralateral forelimb when tail is lifted (mild neurological damage); 2 points, rotation to the contralateral side when crawling (moderate neurological damage); 3 points, falling to the contralateral side when standing or crawling (severe neurological damage); 4 points, no voluntary movement with impaired consciousness. A score of 1 to 4 indicates successful modeling.

[0079] 1.7 Specimen Collection and Processing

[0080] After neurological function assessment, three rats were anesthetized intraperitoneally, and their thoracic cavities were dissected to expose the heart. The right atrial appendage was cut open, and a syringe needle was inserted into the left atrium for fixation. 150 mL of paraformaldehyde was then rapidly injected through the syringe. Once no bloody fluid flowed from the heart, muscle twitching ceased, limbs stiffened, and the forepaws and lungs turned white, the brain was removed via craniotomy and sealed with 50 mL of paraformaldehyde for hematoxylin-eosin (HE) staining and Nissl body staining (NS). Six rats were sacrificed, and their brains were removed via craniotomy. After washing with PBS, the brains were frozen for 30 min, and coronal sections were used for TTC staining. The remaining rats were sacrificed, and their brains were removed. The left ischemic hemisphere was excised and rapidly frozen in liquid nitrogen at -80°C for subsequent indicator detection.

[0081] 1.8 Observation Indicators and Methods

[0082] 1.8.1 TTC Staining for Infarct Volume Frozen rat brains were cut into five sections along the coronal plane, each approximately 2 mm thick. These sections were placed in 2% TTC staining solution and incubated at 37°C in the dark for 30 min. After staining, the sections were fixed with 4% paraformaldehyde and photographed. The volume of the infarcted region was measured using ImageJ image analysis software, and the infarct rate was calculated. Infarct rate = (Infarct volume / Total brain volume) × 100%.

[0083] 1.8.2 HE staining of brain tissue: Brain tissue samples were collected, dehydrated and cleared, impregnated with paraffin, embedded in paraffin, and then cut into thin sections of 3-5 μm. After mounting and dewaxing and dehydration, HE staining was performed, and the sections were mounted. The distribution of neurons, morphology and other pathological changes in the brain tissue were observed under an optical microscope and photographed and preserved.

[0084] 1.8.3 The specific experimental steps for baking, dewaxing and hydrating paraffin sections stained with Nissl stain, staining with Nissl stain, dehydration, clearing, and mounting should be strictly followed according to the instructions for Nissl stain reagent. The sections should be observed and photographed under a microscope.

[0085] 1.8.4 Colorimetric determination of Fe in brain tissue 2+ To determine the levels of MDA, SOD, and GSH, brain tissue samples were collected. 0.05g of brain tissue was weighed from each group, and 0.45mL of physiological saline or PBS was added. The samples were then homogenized for 1 minute using an automatic homogenizer to prepare a 10% homogenate. The homogenate was centrifuged at 3000×g at 4℃, and the supernatant was collected. The procedure was followed according to the instructions of the corresponding kit, and Fe was calculated. 2+ Content of MDA, SOD and GSH.

[0086] 1.8.5 Real-time quantitative PCR was used to detect the expression of SLC7A11, GPX4, and other mRNAs in ischemic brain tissue. Total RNA was extracted from rat ischemic brain tissue according to the RNA extraction kit instructions. The concentration and purity were determined, cDNA was synthesized, and real-time quantitative PCR was performed. The reaction conditions were: 95℃ pre-denaturation for 5 s; PCR reaction, 95℃ for 10 s, 55℃ for 30 s, 40 cycles. The expression of SLC7A11, GPX4, excitatory amino acid transporter 1 (EAAT1, GLAST), glutamate transporter 2 (EAAT2, GLT1), glutaminase (GS), and glutamine synthetase (PAG) mRNAs was detected. GAPDH was used as an internal control. -ΔΔCt Gene expression levels were calculated using the method described in Table 1. The primer sequences were synthesized by Beijing Dingguo Biotechnology Co., Ltd.

[0087] Table 1 Primer sequences

[0088]

[0089] 1.8.6 Western Blot Detection of SLC7A11, GPX4, and FTH1 Protein Expression in Ischemic Brain Tissue Brain tissue samples were collected, and protein extraction was performed. BCA assay was used to calculate the concentration. Electrophoresis was performed at 120V, and the membrane was transferred at 400A for 25 min. After blocking with rapid blocking buffer, primary antibody was added and incubated overnight at 4℃. The primary antibody dilution ratio was SLC7A11 (1:5000), GPX4 (1:10000), FTH1 (1:1000), and GAPDH (1:10000). The membrane was washed 5 times with TBST for 5 min each time. Secondary antibody was then added and incubated at room temperature for 1 h, followed by TBST washing 5 times for 5 min each time. After adding developing buffer, the membrane was scanned using a chemiluminescence image analyzer, and the results were saved. ImageJ software was used to analyze the grayscale values ​​of the target protein bands. GAPDH was used as an internal control protein to calculate the relative expression level of the target protein.

[0090] 1.9 Statistical Methods

[0091] Statistical analysis was performed using SPSS 20.0 software. Experimental data are expressed as mean ± standard deviation. The results indicate that the differences between groups were statistically analyzed using one-way ANOVA. Pairwise comparisons were performed using the LSD test, provided that homogeneity of variance was satisfied. All statistical tests were two-tailed, and p < 0.05 was considered statistically significant.

[0092] 2 Results

[0093] 2.1 Effects of Artemisia annua extract on neurological function scores in IS rats

[0094] The neurological function score in the sham-operated group was 0. Compared with the sham-operated group, the neurological function deficit score in the model group rats was significantly increased (P<0.01). Compared with the model group, the neurological function deficit scores in the Ginkgo biloba extract group, and the Artemisia annua extract groups (25 mg / kg, 50 mg / kg, and 100 mg / kg) were decreased to varying degrees (P<0.05, P<0.01). Results are shown in Table 2 and... Figure 1 .

[0095] Table 2 Neurological deficit scores for each group ( n=6)

[0096] Group score Sham surgery group 0.00±0.00 Model group <![CDATA[3.1±0.75 ** ]]> Ginkgo biloba extract group <![CDATA[2.17±0.75 ## ]]> Artemisia annua extract 12.5 mg / kg 2.5±0.84 Artemisia annua extract 25mg / kg <![CDATA[2.33±0.52 # ]]> Artemisia annua extract 50mg / kg <![CDATA[1.67±0.52 ## ]]> Artemisia annua extract 100mg / kg <![CDATA[1.83±0.75 ## ]]>

[0097] Note: Compared with the sham surgery group. ** P<0.01, compared with the model group, # P<0.05, ## P<0.01.

[0098] 2.2 Effects of Artemisia annua extract on cerebral infarction volume in IS rats

[0099] After TTC staining, normal rat brain tissue appeared rose-red, while ischemic infarcts appeared pale white. No cerebral infarction was observed in the sham-operated group. Compared with the sham-operated group, a distinct white infarct area was visible on the ischemic side in the model group, and the infarct volume was significantly increased (P<0.01). Compared with the model group, the infarct volume was significantly reduced in the Ginkgo biloba extract group, and in the Artemisia annua extract groups (25 mg / kg, 50 mg / kg, and 100 mg / kg) (P<0.05, P<0.01), indicating that Artemisia annua extract can reduce the infarct volume in IS rats. Results are shown in Table 3 and... Figure 2 , 3 .

[0100] Table 3. Infarct volume in rats of each group ( n=6)

[0101]

[0102]

[0103] Note: Compared with the sham surgery group. ** P<0.01, compared with the model group, # P<0.05, ## P<0.01.

[0104] 2.3 Effects of Artemisia annua extract on the pathological changes of hippocampal neurons in ischemic brain tissue of IS rats

[0105] HE staining revealed that hippocampal neurons in the sham-operated group were round, with intact and regular morphology, lightly stained nuclei, and tightly and neatly arranged cells without vacuolated cell bodies. Compared with the sham-operated group, the model group showed a decrease in the number of hippocampal neurons, loose arrangement, abnormal cell morphology, pyknosis of neuronal nuclei, and vacuolation at the periphery. Compared with the model group, the Ginkgo biloba extract group and the Artemisia annua extract groups at doses of 25 mg / kg, 50 mg / kg, and 100 mg / kg showed significant pathological improvement, with increased cell number, relatively regular and intact cell morphology, and compact arrangement. Results are shown below. Figure 4 .

[0106] Nissl bodies are specialized structures of neurons. Nissl staining, using basic dyes to stain nerve tissue, is widely used to observe cellular structures within neurons and to assess neuronal damage. Nissl staining revealed that in the sham-operated group, hippocampal neurons were neatly arranged, structurally clear, and showed uniform Nissl body staining. Compared to the sham-operated group, the model group showed a significant reduction in the number of neurons, uneven arrangement, obvious cell body damage, and unclear Nissl bodies. Compared to the model group, the Ginkgo biloba extract group and the Artemisia annua extract groups at doses of 25 mg / kg, 50 mg / kg, and 100 mg / kg showed a greater distribution of neurons, neat arrangement, intact structure, and clearly visible Nissl bodies. Results are shown below. Figure 5 .

[0107] 2.4 Effects of Artemisia annua extract on Fe in ischemic brain tissue of IS rats 2+ The effects of changes in MDA, GSH and SOD content

[0108] Compared with the sham-operated group, the model group rat brain tissue Fe 2+ The levels of MDA were significantly increased, while the levels of GSH and SOD were significantly decreased (P<0.01). Compared with the model group, the levels of Fe in the brain tissue of rats in the Ginkgo biloba extract group and the Artemisia annua extract groups at doses of 25 mg / kg, 50 mg / kg, and 100 mg / kg were significantly increased. 2+ The content was significantly reduced (P<0.01); the MDA content in the brain tissue of rats in the Ginkgo biloba extract group and the Artemisia annua extract groups at doses of 50 mg / kg and 100 mg / kg were all decreased (P<0.05, P<0.01); the SOD and GSH contents in all treatment groups increased to varying degrees (P<0.05, P<0.01). Results are shown in […]. Figure 6 .

[0109] 2.5 Effects of Artemisia annua extract on the expression levels of SLC7A11, GPX4, EAAT1, EAAT2, GS, and PAG mRNA in ischemic brain tissue of IS rats

[0110] Compared with the sham-operated group, the expression levels of SLC7A11, GPX4, and EAAT2 mRNA in the model group were significantly decreased (P<0.05, P<0.01); compared with the model group, the expression level of SLC7A11 mRNA in the 50 mg / kg and 100 mg / kg dose groups of Artemisia annua extract was significantly increased (P<0.05, P<0.01); the expression level of GPX4 mRNA in the Ginkgo biloba extract group and each dose group of Artemisia annua extract was increased (P<0.05); and the expression level of EAAT2 mRNA in each dose group of Artemisia annua extract was significantly increased (P<0.05, P<0.01).

[0111] Compared with the sham-operated group, the expression levels of EAAT1 and GS mRNA in the model group were slightly increased, but the difference was not statistically significant (P>0.05). Compared with the model group, the expression levels of EAAT1 and GS mRNA in each treatment group were slightly decreased, but the difference was not statistically significant (P>0.05).

[0112] Compared with the sham-operated group, the expression level of PAG mRNA in the model group was significantly increased (P<0.01); compared with the model group, the expression level of PAG mRNA in the Artemisia annua extract treatment groups at doses of 12.5 mg / kg, 50 mg / kg, and 100 mg / kg was significantly decreased (P<0.05). Results are shown below. Figure 7 .

[0113] 2.6 Effects of Artemisia annua extract on the expression of SLC7A11, GPX4, and FTH1 proteins in ischemic brain tissue of IS rats

[0114] Compared with the sham-operated group, the protein expression of SLC7A11, GPX4, and FTH1 in the model group was significantly decreased (P < 0.01). Compared with the model group, the protein expression of SLC7A11 in the Ginkgo biloba extract group and the Artemisia annua extract groups at doses of 12.5 mg / kg, 50 mg / kg, and 100 mg / kg was significantly increased (P < 0.05, P < 0.01); the protein expression of GPX4 in the Artemisia annua extract groups at doses of 25 mg / kg and 50 mg / kg was significantly increased (P < 0.01); and the protein expression of FTH1 in the Ginkgo biloba extract group and the Artemisia annua extract group at dose of 25 mg / kg was significantly increased (P < 0.05). Results are shown below. Figure 8 , 9 .

[0115] 3. Conclusion

[0116] This study found that 24 hours after the onset of ischemic stroke (IS) in rats, the neurological deficit score significantly increased, with symptoms such as hemiplegia, unidirectional spinning, and flexion of the forelimbs to the contralateral side when the tail was lifted. TTC staining revealed large areas of pale ischemic brain tissue. HE staining and Nissl body staining showed a decrease in the number of neurons, loose arrangement, abnormal morphology, vacuolation at cell edges, and blurred Nissl bodies. Rats pretreated with artemisinin extract showed a decrease in neurological deficit score, a reduction in various neurological symptoms, a significantly smaller ischemic area in the brain compared to the model group, less damage to the microstructure, a greater number of cells, intact morphology, tight arrangement, and clear and distinct Nissl bodies. These results indicate that artemisinin extract has a protective effect on the morphology and function of rat brain tissue after the onset of IS, and has positive significance for the prevention and progression of ischemic stroke.

[0117] This study also found that after IS occurred in rats, Fe in the ischemic brain tissue of rats was... 2+ Elevated MDA levels, decreased GSH and SOD levels, decreased expression levels of SLC7A11 and GPX4 genes and proteins, decreased FTH1 protein levels, decreased EAAT2 mRNA expression, and increased PAG mRNA expression indicate that excessive glutamate accumulation in brain tissue after ischemic stroke (IS) induces ferroptosis in neurons. Artemisia annua extract can reduce Fe in ischemic brain tissue of rats. 2+ Artemisia annua extract not only maintains iron homeostasis in brain tissue by increasing MDA levels, raising GSH and SOD levels, upregulating the expression levels of suppressed SLC7A11 and GPX4 genes and proteins, and increasing FTH1 protein expression, but also activates the GSH-GPX4 antioxidant system, which defends against ferroptosis, thereby protecting nerve cells and inhibiting neuronal ferroptosis. Furthermore, Artemisia annua extract upregulates EAAT2 mRNA expression, promotes the transport of excess extracellular glucose to astrocytes, activates the body's self-repair function, and maintains glucose homeostasis. Simultaneously, Artemisia annua extract reduces PAG mRNA expression, decreasing the synthesis of new glucose and further protecting nerve cells.

[0118] In summary, excessive glutamate (Glu) accumulation in the ischemic region of ischemic stroke rats leads to characteristic changes of ferroptosis, such as ferrous ion accumulation and lipid peroxidation. Artemisia annua extract alleviates neuronal cell damage in ischemic stroke rats by regulating glutamate metabolism and inhibiting neuronal ferroptosis, thereby achieving the effect of preventing and / or treating ischemic stroke.

[0119] Example 13: Experimental Study on the Efficacy of Artemisia annua Extract in the Prevention and / or Treatment of Ischemic Stroke (Part Two)

[0120] (I) Determination of Artemisinin Content in Artemisia annua Extract by High Performance Liquid Chromatography

[0121] 1. Instruments and Materials

[0122] 1.1 Instruments

[0123] The Agilent 1260 high-performance liquid chromatograph is equipped with a G7129A autosampler, a G7115A detector, and a G7116A column oven.

[0124] 1.2 Materials

[0125] The Artemisia annua slices used in the experiment were purchased from Beijing Tongrentang Changyang Pharmacy Co., Ltd., batch number: 20210827, and identified by teachers from the Department of Traditional Chinese Medicine, School of Traditional Chinese Medicine, Beijing University of Chinese Medicine. Artemisia annua extract was prepared according to the method in Example 1 and stored at 4℃ for later use. The reference standard was artemisinin (Chengdu Ruifenside Biotechnology Co., Ltd., catalog number: Q-005); methanol (Thermo Fisher Scientific Co., Ltd., catalog number: A452-4), which was used as the chromatographic alcohol.

[0126] 2 Methods

[0127] 2.1 Preparation of test solution

[0128] Accurately weigh 1.0g of Artemisia annua extract, add methanol to a 50mL volumetric flask, sonicate for 30min, shake well, and filter through a microporous membrane (0.22μm) to obtain the final product.

[0129] 2.2 Preparation of Standard Solutions

[0130] Accurately weigh 5 mg of artemisinin reference standard, add methanol to a volumetric flask and dilute to 5 mL. Sonicate for 30 min to dissolve, preparing a stock solution of reference standard at 1 mg / mL. Filter through a microporous membrane (0.22 μm) to obtain the final product.

[0131] 2.3 Chromatographic conditions

[0132] Column: Agilent ZORBAX SB-C 18 (250mm×4.6mm, 5μm); Mobile phase: methanol (A)-pure water (B), gradient elution; Volumetric flow rate: 0.8mL / min; Column temperature: 30℃; Injection volume: 10μL.

[0133] 3 Results

[0134] The artemisinin content in the Artemisia annua extract is 5.42%.

[0135] (II) Experimental study on the efficacy of artemisinin extract in preventing and / or treating ischemic stroke

[0136] 1. Materials and Methods

[0137] 1.1 Animals

[0138] Forty SPF - level healthy male SD rats, 8 - 10 weeks old, with a body weight of 280 ± 30 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animal production license number is: SYXK(Beijing)2022 - 0052. The animals were housed in the Animal Experiment Center of Beijing University of Chinese Medicine, with a 12 - hour day - night cycle, room temperature (20 ± 2)°C, and relative humidity of 50% - 60%. They were allowed to eat and drink freely. They were adaptively fed for 3 days.

[0139] 1.2 Drugs

[0140] The Artemisia annua Chinese herbal pieces used in the experiment were purchased from Beijing Tongrentang Changyang Pharmacy Co., Ltd., batch number: 20210827, and were identified by teachers from the Traditional Chinese Medicine Teaching and Research Section of the School of Traditional Chinese Medicine of Beijing University of Chinese Medicine. They were prepared into Artemisia annua extracts according to the method of Example 1 and stored in a 4°C refrigerator for later use.

[0141] 1.3 Main reagents

[0142] Middle cerebral artery occlusion thrombus line (Beijing Xinuo Technology Co., Ltd., product number: 26 A4); Triphenyltetrazolium chloride (2,3,5 - triphenyltetrazolium chloride, TTC, Beijing Solarbio Science & Technology Co., Ltd., product number: G3005); Artemisinin (Shanghai Yuanye Bio - Technology Co., Ltd., product number: S31425).

[0143] 1.4 Grouping and drug administration

[0144] Forty male SD rats were divided into a sham - operation group, a model group, an artemisinin group, and an Artemisia annua extract group by the random number table method, a total of 4 groups, with 10 rats in each group. In the artemisinin group, according to the content of artemisinin in the Artemisia annua extract determined by high - performance liquid chromatography, the dose of artemisinin corresponding to 50 mg / kg of Artemisia annua extract was calculated to be 2.71 mg / kg. Therefore, the artemisinin group was administered at 2.71 mg / kg, and the Artemisia annua extract group was administered at 50 mg / kg. For each drug - administrated group, the drug was evenly mixed with a 0.5% sodium carboxymethylcellulose (CMC - Na) suspension, and the sham - operation group and the model group were gavaged with a 0.5% CMC - Na suspension. The drugs were administered by gavage once 30 minutes before modeling.

[0145] 1.5 Modeling method

[0146] Preoperative fasting (but water intake permitted) was maintained for 12 hours. A rat model of middle cerebral artery occlusion (MCAO) was established using the suture occlusion method. An anesthetic was prepared using a 1:1 mixture of 5% chloral hydrate and 10% urethane, administered intraperitoneally at a dose of 0.5 mL / 100 g. A 1-2 cm incision was made along the midline of the neck of the deeply anesthetized rat to expose the left common carotid artery. This artery was bluntly dissected from the left fascia, and the internal and external carotid arteries were isolated. The external carotid artery was briefly clamped. After ligating the proximal end of the common carotid artery, a V-shaped incision was made, and a suture was inserted, slightly adjusted to penetrate the internal carotid artery to a depth of approximately 19 mm. The distal end of the incision was then ligated, excess suture was cut, the wound was disinfected with antibiotics, and sutured. The rats were placed on a warming pad for postoperative warming until the anesthesia wore off. The sham surgery group underwent the same procedures as the rats in the sham surgery group, except that no suture was inserted.

[0147] 1.6 Zea Longa scoring method for detecting neurological function in rats

[0148] Neurological deficit assessment was performed 24 hours after modeling. The Zea-Longa neurological deficit assessment scale is a 5-point scale from 0 to 4: 0 points, no neurological damage; 1 point, adduction and flexion of the contralateral forelimb when tail is lifted (mild neurological damage); 2 points, rotation to the contralateral side when crawling (moderate neurological damage); 3 points, falling to the contralateral side when standing or crawling (severe neurological damage); 4 points, no voluntary movement with impaired consciousness. A score of 1 to 4 indicates successful modeling.

[0149] 1.7 TTC staining to detect cerebral infarction volume

[0150] After sacrificing rats, craniotomy was performed to harvest the brain. The brain was washed with PBS and frozen for 30 minutes. The frozen whole brain was then cut into five sections along the coronal plane, each approximately 2 mm thick. These sections were placed in 2% TTC staining solution and incubated at 37°C in the dark for 30 minutes. After staining, the brain was fixed with 4% paraformaldehyde and photographed. ImageJ image analysis software was used to measure the volume of the infarcted area and calculate the infarct rate. The infarct volume ratio was calculated as (infarct volume / total brain volume) × 100%.

[0151] 1.8 Statistical Methods

[0152] Statistical analysis was performed using SPSS 20.0 software. Experimental data are expressed as mean ± standard deviation. The results indicate that the differences between groups were statistically analyzed using one-way ANOVA. Pairwise comparisons were performed using the LSD test, provided that homogeneity of variance was satisfied. All statistical tests were two-tailed, and p < 0.05 was considered statistically significant.

[0153] 2 Results

[0154] 2.1 Effects of artemisinin and artemisia extract on neurological function scores in IS rats

[0155] The neurological function score of rats in the sham-operated group was 0. Compared with the sham-operated group, the neurological function deficit score of rats in the model group was significantly increased (P<0.01). Compared with the model group, the neurological function deficit scores of rats in the artemisinin group and the artemisia annua extract group were decreased to varying degrees (P<0.05, P<0.01). Compared with the artemisinin group, the neurological function score of rats in the artemisia annua extract group was significantly decreased (P<0.05). The results are shown in Table 4.

[0156] Table 4. Neurological deficit scores of rats in each group (x±s, n=10)

[0157] Group score Sham surgery group 0.000±0.000 Model group <![CDATA[2.429±0.535 ** ]]> Artemisinin group <![CDATA[1.857±0.378 # ]]> Artemisia annua extract group <![CDATA[1.333±0.516 ##△ ]]>

[0158] Note: Compared with the sham surgery group. ** P<0.01; compared with the model group, # P<0.05, ## P<0.01; compared with the artemisinin group, △ P<0.05.

[0159] 2.2 Effects of artemisinin and artemisia extract on cerebral infarction volume in IS rats

[0160] After TTC staining, normal rat brain tissue appeared rose-red, while ischemic infarcts appeared pale white. Compared with the sham-operated group, the model group showed a distinct white infarct area on the ischemic side, and the infarct volume was significantly increased (P<0.01). Compared with the model group, the artemisinin and artemisia extract groups showed a significant decrease in infarct volume (P<0.05, P<0.01); compared with the artemisinin group, the artemisia extract group showed a significant decrease in infarct volume (P<0.01). Results are shown in Table 5. Figure 10 .

[0161] Table 5. Cerebral infarction volume in rats of each group (x±s, n=8)

[0162] Group Cerebral infarction volume (%) Sham surgery group 6.366±0.940 Model group <![CDATA[31.050±2.679 ** ]]> Artemisinin group <![CDATA[28.400±2.302 # ]]> Artemisia annua extract <![CDATA[22.769±2.372 ##△△ ]]>

[0163] Note: Compared with the sham surgery group. ** P<0.01; compared with the model group, # P<0.05, ## P<0.01; compared with the artemisinin group, △△ P<0.01.

[0164] Example 14: Study on the protective effect and mechanism of artemisia annua extract against glutamate-induced ferroptosis in glutamate-damaged mouse hippocampal neurons (HT22 cells).

[0165] 1. Protective effect of Artemisia annua extract-containing serum on glutamate-induced HT22 cell damage

[0166] After 6mM glutamate modeling and appropriate treatment, cell viability was detected by CCK-8 assay 24 h later. The results are as follows: Figure 11 As shown, compared with the normal control group, the survival rate of HT22 cells in the model group was significantly reduced (P<0.01); compared with the model group, the cell survival rates of Liproxstatin-1 (Lip-1) and Artemisia annua extract groups were significantly increased (P<0.01), indicating that Artemisia annua extract can improve the survival rate of glutamate-induced HT22 cells and play a protective role against glutamate-induced ferroptosis in HT22 cells.

[0167] 2. Effects of Artemisia annua extract on the levels of MDA, GSH, and SOD in glutamate-induced HT22 cells

[0168] When cells undergo ferroptosis, lipid peroxidation occurs, with MDA being a key end product. MDA levels can be used to describe the degree of lipid peroxidation. GSH levels indicate the normal functioning of the cellular ferroptosis defense pathway, while SOD is an intracellular enzyme used to scavenge oxygen free radicals.

[0169] The results of the MDA content detection experiment showed that, compared with the normal control group, the MDA content of HT22 cells treated with 6mM glutamate was significantly increased (P<0.01). Compared with the model group, the MDA levels of the Lip-1 group and the high-dose Artemisia annua extract group were decreased (P<0.05, P<0.01). Figure 12 A. GSH level detection results showed that, compared with the normal control group, the GSH content of HT22 cells treated with 6mM glutamate was significantly reduced (P<0.01). Compared with the model group, the GSH content of the Lip-1 group and the medium and high dose groups of Artemisia annua extract was significantly increased (P<0.05, P<0.01). Figure 12 B. SOD content detection results showed that compared with the normal control group, the SOD content in the model group was significantly decreased (P<0.01), and compared with the model group, the SOD content in each treatment group was significantly increased (P<0.05, P<0.01). The results are shown in [the table below]. Figure 12 C.

[0170] Based on the above experimental results, it is further demonstrated that glutamate-induced ferroptosis occurred in HT22 cells, and that artemisia annua extract exerted an anti-glutamate-induced ferroptosis effect on HT22 cells by reducing the content of MDA and increasing the content of GSH and SOD.

[0171] 3. Effects of Artemisia annua extract on the expression levels of SLC7A11 and GPX4 mRNA in HT22 cells

[0172] The GSH-GPX4 antioxidant system is the most classic and important defense system against ferroptosis, and SLC7A11 and GPX4 are key regulatory proteins in the GSH-GPX4 antioxidant system. This experiment assessed the antioxidant capacity and protective effect of artemisia annua extract on HT22 cells after glutamate damage by detecting the expression levels of SLC7A11 and GPX4 mRNA.

[0173] like Figure 13 As shown in Figure A, compared with the normal control group, the expression level of SLC7A11 mRNA in the model group decreased after glutamate treatment (P < 0.05). Compared with the model group, the expression level of SLC7A11 mRNA in the Lip-1 group and each dose group of Artemisia annua extract increased significantly (P < 0.05, P < 0.01).

[0174] like Figure 13 As shown in B, compared with the normal control group, the expression level of GPX4 mRNA in the model group was significantly decreased after glutamate treatment (P<0.05). Compared with the model group, the expression level of GPX4 mRNA in the Liproxstatin-1 group and each dose group of Artemisia annua extract was significantly increased (P<0.01).

[0175] The results showed that after glutamate toxicity damage, the mRNA expression levels of SLC7A11 and GPX4 in HT22 cells decreased significantly, impairing cellular antioxidant capacity and leading to ferroptosis. Artemisia annua extract can increase the mRNA expression levels of SLC7A11 and GPX4, helping to restore antioxidant capacity, defend against ferroptosis, and protect nerve cells from damage.

[0176] 4. Western blot analysis of the effects of Artemisia annua extract on the expression of SLC7A11, GPX4 and FTH1 proteins in rats.

[0177] like Figure 14 As shown in A and 14B, compared with the normal control group, the expression level of SLC7A11 protein in the model group decreased significantly after glutamate treatment (P < 0.05); compared with the model group, the expression level of SLC7A11 protein in the Lip-1 group and each dose group of Artemisia annua extract increased significantly (P < 0.01).

[0178] like Figure 14 As shown in A and 14C, compared with the normal control group, the expression level of GPX4 protein in the model group was significantly reduced after glutamate treatment (P < 0.05); compared with the model group, the expression level of GPX4 protein in the Lip-1 group and each dose group of Artemisia annua extract was significantly increased (P < 0.05).

[0179] like Figure 14As shown in A and 14D, compared with the normal control group, the FTH1 protein level in the model group showed a decreasing trend after glutamate treatment, but the difference was not statistically significant (P>0.05). Compared with the model group, the FTH1 protein expression level in the Lip-1 group and each dose group of Artemisia annua extract showed an increasing trend, but the difference was not statistically significant (P>0.05).

[0180] In summary, cell experiments showed that the gene and protein expression of SLC7A11 and GPX4 in HT22 cells was inhibited after glutamate treatment, while Artemisia annua extract could defend against ferroptosis by increasing the gene and protein expression levels of SLC7A11 and GPX4.

Claims

1. Application of Artemisia annua extract in the preparation of drugs for the prevention and / or treatment of ischemic stroke.

2. The application as described in claim 1, characterized in that, The application of artemisinin extract in regulating glutamate metabolism.

3. The application as described in claim 1, characterized in that, The application of artemisinin extract in inhibiting neuronal ferroptosis.

4. The application as described in any one of claims 1-3, characterized in that, The artemisia extract is an organic solvent extract of artemisia.

5. The application as described in claim 4, characterized in that, The organic solvent is selected from any one or more of methanol, ethanol, n-butanol, acetone, ethyl acetate, chloroform, and petroleum ether.

6. The application as described in claim 5, characterized in that, The organic solvent is methanol.

7. The use of artemisinin in the preparation of drugs for the prevention and / or treatment of ischemic stroke.

8. A composition for the prevention and / or treatment of ischemic stroke, characterized in that, The composition comprises an effective amount of artemisia annua extract and / or artemisinin, and a pharmaceutically acceptable carrier.

9. The composition according to claim 8, characterized in that, The Artemisia annua extract is an organic solvent extract of Artemisia annua; the organic solvent is selected from any one or more of methanol, ethanol, n-butanol, acetone, ethyl acetate, chloroform, and petroleum ether.

10. The composition according to claim 8, characterized in that, The composition is prepared into injections, capsules, tablets, granules, gels, sustained-release preparations, oral liquids, pellets, or nano-preparations.

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