Use of alpha-asarone in the manufacture of a drug for the prevention or treatment of hemorrhagic stroke

Abstract

The present invention provides the use of α-asarone in the manufacture of a drug for preventing or treating hemorrhagic stroke, the structure of α-asarone is shown in formula I, which significantly improves short-term neurological function deficit and long-term learning memory function in model rats, relieves cerebral edema, improves blood-brain barrier permeability, and prevents or alleviates cerebral tissue atrophy in the recovery period, has obvious therapeutic effect on animal models of hemorrhagic stroke, has no obvious toxicity and side effects, and is expected to be a preventive / treatment drug for hemorrhagic stroke. [Formula 1] JPEG2024512842000012.jpg31163

Description

[Technical Field]

[0001] The present invention belongs to the field of biomedicine and relates to the use of alpha-asarone in the manufacture of a medicament for the treatment or prevention of hemorrhagic stroke. [Background technology]

[0002] Stroke is currently the second leading cause of death in the world, with nearly 4 million new cases occurring annually in China alone, the highest incidence rate in the world. More than 2 million people die from stroke each year, and approximately two-thirds of surviving stroke patients are permanently disabled. Clinically, strokes are classified as ischemic stroke and hemorrhagic stroke. Hemorrhagic stroke occurs when a blood vessel inside the skull ruptures, causing blood to leak into the brain, resulting in various clinical symptoms, including nervous system dysfunction. Hemorrhagic stroke has a relatively low incidence rate but high mortality and disability rates. Hemorrhagic stroke is divided into two types based on the location of bleeding within the brain tissue: intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). ICH occurs intracerebrally, while SAH occurs between the leptomeninges and arachnoid membrane. Hypertensive intracerebral hemorrhage is the most common cause of non-traumatic ICH, whereas intracranial aneurysms are a common cause of SAH.

[0003] Brain injury from hemorrhagic stroke poses a clinical challenge and is a significant cause of disability. Brain injury can be divided into two types: primary and secondary. Primary brain injury refers to direct mechanical compression and ischemic changes in the surrounding brain tissue caused by the hematoma resulting from the initial hemorrhage and its expansion, including glutamate overload, calcium overload, and mitochondrial dysfunction. Secondary brain injury has a more complex mechanism, with pathological pathways including blood-brain barrier disruption, cerebral edema formation, oxidative stress and inflammatory responses, autophagy and apoptosis, microglial activation, changes in brain energy metabolism and proteome, and iron deposition, ultimately leading to neurological impairment. The pathological mechanisms of hemorrhagic stroke brain injury involve a variety of factors and links, many of which interact and are interconnected. Among these, neuronal excitotoxicity due to imbalanced regulation of excitatory amino acids (e.g., glutamate) and inhibitory amino acids (e.g., gamma-aminobutyric acid, GABA) is the main cause of neuronal damage and death in the acute phase of hemorrhagic stroke.

[0004] Currently, clinical treatment for hemorrhagic stroke patients mainly involves drug therapy and surgical treatment. Drug therapy is primarily symptomatic medical treatment, including intracranial pressure reduction, blood pressure regulation, hemostatic therapy, hypothermia, cerebral metabolic stimulants, and calcium channel blockers, but is ineffective. While surgical treatment actually plays a positive role in saving patients' lives, its effectiveness in treating neurological dysfunction is less than ideal and it has more stringent application requirements. To date, no drug therapy has been approved to treat neurological damage caused by hemorrhagic stroke, thereby increasing patient survival or improving patient prognosis. Therefore, developing drugs that can effectively treat hemorrhagic stroke is of great clinical importance.

[0005] α-Asarone is the main active ingredient of the Chinese herbal medicine Acorus calamus, and has sedative, antispasmodic, and anticonvulsant properties. Research has shown that α-asarone blocks Na+ channels and inhibits GABA AIt has been shown that activating the GABA receptor can exert antiepileptic effects (Wang ZJ, Levinson SR, Sun L, et al. Identification of both GABA receptors A receptors and voltage-activated Na + (See also "Channels as Molecular Targets of the Anticonvulsant α-Asarone[J]. Front Pharmacol, 2014, 5(40):5-11" and "Huang C, Li WG, Zhang XB, et al. α-Asarone from Acorus gramineus Alleviates Epilepsy by Modulating A-Type GABA Receptors[J]. Neuropharmacology, 2013, 65(2):1-11.") It also promotes proliferation of neural progenitor cells, reduces oxidative stress, inhibits microglial activation, suppresses neuroinflammation, and ameliorates neuronal apoptosis. (See also "Chellian R, Pandy V, Mohamed Z. Pharmacology and Toxicology of α- and β-Asarone: A Review of Preclinical Evidence[J]. Phytomedicine, 2017, 41-58.") Although the above studies suggest that α-asarone has multiple neuropharmacological activities, the therapeutic effect of α-asarone on hemorrhagic stroke has not been reported to date.

[0006] On the other hand, in the clinical treatment of secondary epilepsy caused by hemorrhagic stroke, the prophylactic administration of antiepileptic drugs is generally not recommended (Chinese Medical Association Neurological Society, Chinese Medical Association Neurological Cerebrovascular Disease Group, Chinese Cerebral Hemorrhage Diagnosis and Treatment Guidelines 2019[J]. Chinese Journal of Neurology, 2019, 52(12):994-1005.). This is because antiepileptic drugs have a high incidence of side effects, and the prophylactic administration of antiepileptic drugs may impair the neurological function of patients with hemorrhagic stroke. Summary of the Invention

[0007] To overcome the lack of drugs for the prevention or treatment of hemorrhagic stroke in the prior art, the present invention provides a novel use of alpha-asarone.

[0008] For this reason, the present invention provides the following technical solutions:

[0009] The present invention provides the use of a compound of formula I (trans-2,4,5-trimethoxy-1-propenylbenzene, also known as α-asarone) in the manufacture of a medicament for the prevention or treatment of hemorrhagic stroke. [ka] The present invention has unexpectedly found that the compound of formula I can significantly improve short-term neurological function deficits and long-term learning and memory function in rat models, reduce cerebral edema, improve blood-brain barrier permeability, and prevent or alleviate brain tissue atrophy during recovery, and has a clear therapeutic effect on animal models of hemorrhagic stroke, without obvious toxicity and side effects.The present invention has found that by using α-asarone, the positive drug vinpocetine injection and nimodipine injection in the treatment of rat models of subarachnoid hemorrhage established by intravascular puncture and rat models of cerebral parenchymal hemorrhage established by collagenase injection, α-asarone can significantly reduce the edema of the affected brain tissue in the model rats, improve blood-brain barrier permeability, prevent or alleviate brain tissue atrophy during recovery, and significantly improve short-term neurological function scores and long-term learning and memory function.In addition, α-asarone can significantly reduce the incidence and mortality rate of secondary epilepsy caused by acute hemorrhagic stroke in rat models, and prolong survival time.

[0010] In some embodiments, the medicament is further used to prevent or treat secondary epilepsy due to hemorrhagic stroke. Preferably, the medicament is used for the treatment of hemorrhagic stroke and the prevention of secondary epilepsy due to hemorrhagic stroke.

[0011] In some embodiments, the hemorrhagic stroke is a stroke caused by at least one of intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH).

[0012] In the present invention, the compound represented by formula I has the following pharmacological actions: (1) antagonizing neuroexcitotoxicity caused by excess glutamate, (2) suppressing abnormal increases in glutamate and GABA, (3) inhibiting calcium influx into neurons and suppressing intracellular calcium overload, (4) stabilizing the mitochondrial membrane potential of neurons and suppressing apoptosis of neurons, and (5) alleviating oxidative stress responses in damaged neurons.

[0013] In the present invention, the drug has the following pharmacological effects: (1) antagonizing neuroexcitotoxicity caused by excess glutamate, (2) suppressing abnormal increases in glutamate and GABA levels, (3) inhibiting calcium influx in neurons and suppressing intracellular calcium overload, (4) stabilizing the mitochondrial membrane potential of neurons and suppressing apoptosis of neurons, and (5) alleviating oxidative stress responses in damaged neurons.

[0014] In the present invention, the drug has the following functions: (1) antagonizing glutamate excitotoxicity caused by cerebral hemorrhage by reducing the amount of glutamate in the brain of model rats; (2) restoring GABA levels and promoting recovery of motor function in model rats; (3) reducing Ca 2+ Inhibits Ca inflow 2+ (4) alleviate harmful biochemical reactions and excitotoxicity caused by overload; (5) stabilize mitochondrial membrane potential and inhibit neuronal apoptosis; (6) reduce oxidative stress and damage to neurons, thereby alleviating cerebral edema, improving blood-brain barrier permeability, preventing or alleviating brain tissue atrophy during the recovery period, improving short-term neurological function deficits and long-term learning and memory dysfunction in model rats, and exerting anti-hemorrhagic stroke effects.

[0015] In some embodiments, the drug is used for at least one of the following: ameliorating neurological or motor function damage (e.g., neurological or motor function damage due to ICH or SAH), reducing secondary early brain damage (e.g., acute brain tissue edema or blood-brain barrier dysfunction, e.g., acute brain tissue edema or blood-brain barrier dysfunction due to ICH or SAH), reducing acute mortality due to hemorrhagic stroke, extending survival, improving long-term learning and memory impairment due to cerebral hemorrhage, and preventing or alleviating brain tissue atrophy during the recovery period from hemorrhagic stroke.

[0016] In some embodiments, the compound of Formula I is the only active ingredient of the medicament.

[0017] In some embodiments, the drug may contain pharmaceutical adjuvants. Preferably, the total weight ratio of the compound represented by Formula I to the pharmaceutical adjuvants is 1:20 to 1000, for example, 1:20 to 200. More preferably, the compound represented by Formula I is the only active ingredient of the drug, and the total weight ratio of the compound represented by Formula I to the pharmaceutical adjuvants is 1:20 to 1000, for example, 1:20 to 200.

[0018] In some embodiments, the subject of administration of the drug may be a human or an animal. When the drug is used to treat a rat model of hemorrhagic stroke, the effective daily dose of the compound represented by Formula I in the drug can be 5 mg to 40 mg / kg body weight. When the drug is used to treat a human suffering from hemorrhagic stroke, the daily dose range of the compound represented by Formula I in the drug can be 0.15 mg to 5.0 mg / kg body weight, preferably 0.3 mg to 3.0 mg / kg body weight. For example, the drug is administered two to three times a day, with a single dose ranging from 0.15 mg to 1.5 mg / kg body weight, preferably 0.3 mg to 1.5 mg / kg body weight. The above doses can be calculated according to the dose conversion relationships between different animal species.

[0019] In some embodiments, the route of administration of the drug is injection, oral administration, subcutaneous implantation, inhalation, transdermal administration, transmucosal administration, etc. Preferably, the route of administration of the drug is injection (preferably intravenous injection) or oral administration.

[0020] In the present invention, the drug may be in any dosage form suitable for human and / or animal use, for example, any dosage form suitable for different administration routes, as long as the drug is in a dosage form that allows the compound of formula I to enter the brain and reach a therapeutically effective concentration. In some embodiments, the drug is an emulsion (e.g., emulsion injection, oral emulsion). Emulsions are safer than currently available injections (solution-type injections) and have higher bioavailability than tablets.

[0021] In some embodiments, the emulsion may comprise a compound of Formula I, a pharmaceutically acceptable oil, a pharmaceutically acceptable emulsifier, and water.

[0022] wherein the pharmaceutically acceptable oil may comprise at least one of soybean oil, medium chain oil, olive oil, and fish oil.

[0023] Here, the pharmaceutically acceptable emulsifier may be at least one of egg yolk lecithin, soybean lecithin, Pluronic® F-68, and polyethylene glycol stearate-15 (Solutol HS15).

[0024] Here, the water may be water for injection or purified water.

[0025] Depending on the emulsifying properties, the emulsion may contain at least one of oleic acid and sodium oleate. The formulation is carried out by dissolving oleic acid in the oil phase and sodium oleate in the aqueous phase, but a mixture of both may also be dissolved in the oil phase and the aqueous phase, respectively.

[0026] Here, the emulsion may further contain glycerol.

[0027] The emulsion may further contain an antioxidant, such as sodium bisulfite, vitamin E, or pyrogallic acid ester.

[0028] For oral administration, the emulsion may contain at least one other suitable additive, such as a preservative or a flavoring agent. The preservative may be a preservative commonly used in the art, such as benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, ethylparaben, propyl esters, and butyl esters. The flavoring agent may be a flavoring commonly used in the art, such as a sweetener, flavoring agent, mucilage, or foaming agent. The sweetener may be simple syrup, stevioside, aspartame, or the like. The flavoring agent may be a fruit flavor such as apple flavor or strawberry flavor. The mucilage may be gelatin, methylcellulose mastic, or the like. The foaming agent may be a mixture of citric acid, tartaric acid, and sodium bicarbonate.

[0029] In some embodiments, the emulsion may contain, by weight, 0.5% to 5% of the compound of Formula I, 5% to 30% of a pharmaceutically acceptable oil, 0.6% to 1.8% of an emulsifier, 0% to 2.5% of glycerol, and the remainder water (e.g., purified water or water for injection). The concentration of the compound of Formula I in the emulsion can vary within a range, and the range of concentration variation depends on the dose, the dose volume, and the solubility of the compound of Formula I in the oil phase.

[0030] In some embodiments, the emulsion is an emulsion injection solution, preferably, in which the total weight ratio of the compound of Formula I to pharmaceutical adjuvants (including water for injection) is 1:20 to 1000, for example, 1:20 to 200.

[0031] wherein the method for preparing the emulsion may include the steps of: mixing a compound of Formula I, a pharmaceutically acceptable oil, a pharmaceutically acceptable emulsifier, and water under high shear to obtain colostrum; and homogenizing the colostrum under high pressure to obtain the emulsion.

[0032] In some embodiments, the method of making the emulsion may include the following steps: Step 1: Under the protection of nitrogen or an inert gas, the compound of formula I is dissolved in a pharmaceutically acceptable oil at 60-80°C to obtain an oil phase, and then an emulsifier and glycerin are dissolved or dispersed in water at 60-80°C to obtain an aqueous phase. Alternatively, under the protection of nitrogen or an inert gas, the compound of formula I and an emulsifier are dissolved or dispersed in a pharmaceutically acceptable oil at 60-80°C to obtain an oil phase, and then glycerin is dissolved in water at 60-80°C to obtain an aqueous phase.

[0033] Step 2: The oil phase and the water phase are mixed under high-speed shear to disperse the oil phase in the water phase and obtain colostrum.

[0034] Step 3: The colostrum is homogenized under high pressure (e.g., 1 to 3 times) until the average droplet size is no greater than 0.5 μm. The colostrum is then filtered and filled into pharmaceutical containers such as glass ampoules, infusion bottles, vials, and soft bags under nitrogen or inert gas protection. Depending on the administration route, emulsions are obtained by rotary autoclaving or adding preservatives without sterilization.

[0035] The shear rate of the high shear may be a conventional shear rate used in the art for small-scale testing or large-scale production of emulsions, for example, 10,000 to 20,000 r·min for small-scale laboratory testing. -1 For example, for large-scale production, the speed may be 2000 to 4000 r·min -1 The actual shear rate depends on the shear radius, and both determine the magnitude of the shear force.

[0036] The shearing time of the high-speed shearing may be a conventional shearing time used in the art for producing emulsions, and may be, for example, 3 to 10 minutes, or, for example, 5 to 8 minutes.

[0037] The homogenization pressure of the high-pressure homogenization may be a conventional homogenization pressure used in the production of emulsions in the art, and may be, for example, 500 to 1500 bar, and is, for example, 500 to 1000 bar.

[0038] The number of cycles of the high-pressure homogenization may be a conventional number of cycles used in the art for producing emulsions, and may be, for example, 1 to 3 times.

[0039] The present invention also provides a pharmaceutical composition for the prevention or treatment of hemorrhagic stroke, wherein said pharmaceutical composition comprises a compound of formula I and pharmaceutical auxiliaries.

[0040] In some embodiments, the pharmaceutical composition is further used to prevent or treat epilepsy secondary to hemorrhagic stroke.

[0041] In some embodiments, the pharmaceutical compositions are used to treat hemorrhagic stroke and to prevent secondary epilepsy resulting from hemorrhagic stroke.

[0042] In some embodiments, the compound of Formula I is the only active ingredient in the pharmaceutical composition.

[0043] In some embodiments, the pharmaceutical composition is an emulsion.

[0044] The present invention also provides a method for treating or preventing hemorrhagic stroke in a subject, comprising administering to said subject a therapeutically or prophylactically effective amount of a compound of formula I.

[0045] Preferably, the method is used to treat or prevent hemorrhagic stroke in a subject, and to treat or prevent epilepsy secondary to hemorrhagic stroke.

[0046] More preferably, the method is used to treat hemorrhagic stroke in a subject and to prevent epilepsy secondary to hemorrhagic stroke.

[0047] Definitions and Explanations Unless otherwise specified, the following terms and phrases used herein have the following meanings. Certain terms or phrases should not be considered indefinite or unclear without a specific definition, but should be understood in their ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0048] Unless otherwise specified, as used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0049] Unless otherwise specified, for purposes of this invention, the term "pharmaceutically acceptable amount" refers to an amount of those compounds, materials, compositions and / or dosage forms that, within the scope of sound medical judgment, is commensurate with a reasonable benefit / risk ratio without undue toxicity, irritation, allergic response, or other problem or complication.

[0050] Unless otherwise specified, the term "pharmaceutical adjuvants" refers to excipients and additives used in the manufacture of pharmaceuticals or the preparation of prescriptions, and is any substance contained in a drug formulation other than the active ingredient. See Part 4 of the Pharmacopoeia of the People's Republic of China (2020 Edition) or Handbook of Pharmaceutical Excipients (Raymond C Rowe, 2009 Sixth Edition).

[0051] Unless otherwise specified, the term "treatment" refers to therapeutic therapy. With respect to a particular disorder, treatment refers to (1) alleviating one or more biological symptoms of a disease or condition, (2) interfering with (a) one or more points in the biological cascade that leads to or causes the condition, or (b) one or more biological symptoms of the condition, (3) ameliorating one or more symptoms, effects, or side effects associated with the condition, or one or more symptoms, effects, or side effects associated with the condition or its treatment, or (4) slowing the progression of the disease or one or more biological symptoms of the condition.

[0052] Unless otherwise specified, the term "prevention" refers to a reduction in the risk of acquiring or developing a disease, disorder, or condition.

[0053] Unless otherwise specified, the term "therapeutically effective amount" refers to an amount of a compound that, when administered to a subject, is sufficient to treat a disease or condition described herein. The "therapeutically effective amount" varies depending on the compound, the condition and its severity, and the age of the patient being treated, but can be adjusted as needed by one skilled in the art. The effective amount also varies depending on the subject (e.g., human or animal) to which it is administered.

[0054] Unless otherwise specified, the term "prophylactically effective amount" refers to an amount sufficient to prevent a disease, disorder, or condition, or to prevent one or more symptoms associated with a disease, disorder, or condition, or to prevent the recurrence of a disease, disorder, or condition.

[0055] Unless otherwise specified, the term "subject" refers to any animal to which a compound according to an embodiment of the present invention is or has been administered, preferably a mammal, and most preferably a human. The term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, with humans being most preferred.

[0056] In the present invention, unless the reaction temperature is specified, the reaction temperature is room temperature, which is usually 20 to 35°C.

[0057] Unless otherwise specified, "secondary epilepsy due to hemorrhagic stroke" in the present invention refers to epileptic seizures secondary to hemorrhagic stroke (excluding lesions unrelated to hemorrhagic stroke) in patients with no history of epilepsy.

[0058] The above preferred conditions can be arbitrarily combined to obtain preferred examples of the present invention, provided that this does not violate common knowledge in the art.

[0059] All reagents and raw materials used in the present invention are commercially available.

[0060] The positive and inventive effects of the present invention include: This invention is the first to disclose that α-asarone has the therapeutic / preventive effect on hemorrhagic stroke. The research results of the pharmacodynamic mechanism show that α-asarone (1) antagonizes glutamate excitotoxicity caused by cerebral hemorrhage by reducing the content of glutamate in the brain of model rats, (2) restores GABA levels and promotes the recovery of motor function in model rats, and (3) reduces Ca 2+ Reduce inflow and Ca 2+ (4) alleviate harmful biochemical reactions and excitotoxicity caused by neuronal overload; (5) stabilize mitochondrial membrane potential and reduce neuronal apoptosis; (6) reduce oxidative stress and damage to neurons, thereby reducing cerebral edema, alleviating brain damage, improving blood-brain barrier permeability, and preventing or alleviating brain tissue atrophy during the recovery period; and further improving short-term neurological function deficits and long-term learning and memory dysfunction in model rats, significantly reducing the incidence and mortality of epilepsy in the acute phase of model rats, prolonging survival time, improving survival rate, and improving prognosis, and exerting the effect of anti-hemorrhagic stroke.

[0061] The present invention unexpectedly discovered that α-asarone is significantly more effective than vinpocetine injection in improving neurological function deficits in rats in the acute stage of SAH, is significantly more effective than nimodipine injection in improving learning and memory function and preventing or alleviating brain tissue atrophy in rats in the recovery stage of SAH, and is significantly more effective than nimodipine injection and significantly more effective than vinpocetine injection in improving neurological function deficits in ICH rats.Therefore, α-asarone is expected to be a drug for preventing / treating hemorrhagic stroke.

[0062] α-Asarone is safe and effective, and no obvious toxicity or side effects of α-asarone were observed in the entire experimental process of the present invention. [Brief explanation of the drawings]

[0063] [Figure 1] Figure 1: Effects of α-asarone on learning and memory function and brain tissue atrophy during recovery in SAH rats. A: Latency to find the platform during acquisition training for each rat group. B: Time spent in the target quadrant and swimming speed for each rat group during spatial exploration. C: Heat maps of activity during spatial exploration for each rat group. The platform location is indicated by a circle, and the quadrant in which it is located is the target quadrant. D: Brains were harvested by cardiac perfusion after the water maze experiment, and the brain tissue atrophy status for each group was observed. P, S, M, and N represent the sham-operated group, SAH model group, medium-dose α-asarone group, and nimodipine-injected group, respectively. Compared to the sham-operated group, ###P<0.001, ##P<0.01, and #P<0.05. Compared to the model group, **P<0.01 and *P<0.05. Compared to the medium-dose α-asarone group, &&&P<0.001 and &&P<0.01. [Figure 2]Figure 2: Effects of α-asarone on brain edema and blood-brain barrier permeability in rat models. A: Water content in different regions of brain tissue from SAH rats in each group. B: Amount of Evans blue exudation from brain tissue from SAH rats in each group. C: Water content in different regions of brain tissue from ICH rats in each group. D: Amount of Evans blue exudation from brain tissue from ICH rats in each group. P, S, I, and M represent the sham-operated group, SAH model group, ICH model group, and α-asarone medium-dose group, respectively. Compared to the sham-operated group, ###P<0.001, ##P<0.01, #P<0.05. Compared to the model group, *P<0.05. [Figure 3] Figure 3: The effect of α-asarone on glutamate and GABA content in brain tissue of model rats. A: Glutamate content in brain tissue of SAH rats in each group. B: GABA content in brain tissue of SAH rats in each group. C: Glutamate content in brain tissue surrounding the hematoma of ICH rats in each group. D: GABA content in brain tissue surrounding the hematoma of ICH rats in each group. Compared to the sham-operated group, ##P<0.01, #P<0.05. Compared to the model group, *P<0.05. [Figure 4] Figure 4: The effect of α-asarone on calcium ion and mitochondrial membrane potential in model rat brain tissue. A: Measurement of calcium ion levels in brain tissue from SAH rats of each group. B: Measurement of mitochondrial membrane potential in brain tissue from SAH rats of each group. C: Statistical graph of calcium ion and mitochondrial membrane potential fluorescence intensity in brain tissue from SAH rats of each group. D: Measurement of calcium ion levels in brain tissue from ICH rats of each group. E: Measurement of mitochondrial membrane potential in brain tissue from ICH rats of each group. F: Statistical graph of calcium ion and mitochondrial membrane potential fluorescence intensity in brain tissue from ICH rats of each group. Compared to the sham-operated group, ###P<0.001, ##P<0.01, #P<0.05. Compared to the model group, ***P<0.001, **P<0.01. [Figure 5]Figure 5: The effects of different doses of α-asarone on PC12 cells injured with 6 μM oxyhemoglobin. Compared with the control group, ###P<0.001, ##P<0.01; compared with the model group, ***P<0.001, **P<0.01. DETAILED DESCRIPTION OF THE INVENTION

[0064] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. In the following examples, experimental methods without specific conditions are selected according to conventional methods and conditions or according to the product instructions.

[0065] Preparation Example 1 Preparation of α-asarone injectable emulsion 0.50-50.0 g of α-asarone and 50.0-300.0 g of soybean oil for injection were weighed and placed in a suitable container. The mixture was heated to 60-80°C under nitrogen gas protection and stirred to dissolve. Subsequently, 6.0-18.0 g of egg yolk lecithin was weighed, added, and stirred to dissolve (0.10-0.50 g of oleic acid, sodium oleate, or a mixture of both was added, if necessary), to prepare an oil phase. Additionally, 0-3.0 g of Pluronic® (F68) and 0-25.0 g of glycerol were weighed, and approximately 800 mL of water was weighed. The mixture was heated to 60-80°C under nitrogen gas protection and stirred to dissolve, forming an aqueous phase. The oil phase was added to the aqueous phase, sheared at high speed for 5-15 minutes, and then water was added to a total volume of 1000 mL to prepare colostrum. The colostrum was then homogenized 1 to 3 times using a high-pressure homogenizer until the average particle size of the homogenized milk droplets became 0.5 μm or less. The homogenized colostrum was then filtered through a filter membrane, and the filtrate was filled into 5 mL to 20 mL glass ampoules under nitrogen gas protection and sterilized using a rotary hot press at 121°C for 8 to 12 minutes to obtain α-asarone injection emulsions containing α-asarone at concentrations of 0.5 to 50 mg / mL.

[0066] Preparation Example 2 Preparation of α-asarone injectable emulsion 10.0 g of α-asarone, 50.0 g of soybean oil for injection, and 50.0 g of medium-chain triglyceride (MCT) for injection were weighed and placed in a suitable container. Under nitrogen gas protection, the mixture was heated to 60-80°C and stirred to dissolve. Next, 12.0 g of egg yolk lecithin and 0.3 g of sodium oleate were weighed, added, and stirred to dissolve, producing an oil phase. 22.0 g of glycerol was weighed, and approximately 800 mL of water was weighed. Under nitrogen gas protection, the mixture was heated to 60-80°C and stirred to dissolve, producing an aqueous phase. The oil phase was added to the aqueous phase, sheared at high speed for 5-15 minutes, and water was added to make a total of 1000 mL to produce colostrum. The colostrum was then homogenized 1 to 3 times using a high-pressure homogenizer until the average particle size of the homogenized milk droplets became 0.5 μm or less. The homogenized colostrum was then filtered through a filter membrane, and the filtrate was filled into 5 mL or 10 mL glass ampoules under nitrogen gas protection and sterilized using a rotary hot press at 121°C for 8 minutes to obtain an α-asarone injection emulsion containing α-asarone at a concentration of 10 mg / mL.

[0067] Preparation Example 3 Preparation of α-asarone injectable emulsion 20.0 g of α-asarone, 100.0 g of soybean oil for injection, and 100.0 g of medium-chain triglyceride (MCT) for injection were weighed and placed in a suitable container. Under nitrogen gas protection, the mixture was heated to 60-80°C and stirred to dissolve. Next, 12.0 g of egg yolk lecithin and 0.3 g of oleic acid were weighed and added to the container. The mixture was stirred and dissolved to prepare the oil phase. 22.0 g of glycerol was weighed and approximately 800 mL of water was weighed and heated to 60-80°C under nitrogen gas protection. The mixture was stirred and dissolved to prepare the aqueous phase. The oil phase was added to the aqueous phase and sheared at high speed for 5-15 minutes. Water was added to the mixture to make a total of 1000 mL to produce colostrum. The colostrum was homogenized 1 to 3 times using a high-pressure homogenizer until the average particle size of the homogenized milk droplets became 0.5 μm or less. The homogenized colostrum was then filtered through a filter membrane. The filtrate was filled into 5 mL or 10 mL glass ampoules under nitrogen gas protection and sterilized using a rotary hot press at 121°C for 8 minutes to obtain an α-asarone injection emulsion containing α-asarone at a concentration of 20 mg / mL.

[0068] Preparation Example 4 Preparation of α-asarone injectable emulsion 1.0 g of α-asarone and 100.0 g of soybean oil for injection were weighed and placed in a suitable container. Under nitrogen gas protection, the mixture was heated to 60-80°C and stirred to dissolve. Next, 12.0 g of egg yolk lecithin and 0.3 g of oleic acid were weighed and added to the container. The mixture was stirred and dissolved to prepare an oil phase. 22.0 g of glycerol was weighed and approximately 800 mL of water was weighed and heated to 60-80°C under nitrogen gas protection. The mixture was stirred and dissolved to prepare an aqueous phase. The oil phase was added to the aqueous phase and sheared at high speed for 5-15 minutes. Water was added to the mixture to make a total of 1000 mL to produce colostrum. The colostrum was homogenized 1 to 3 times using a high-pressure homogenizer until the average particle size of the homogenized milk droplets became 0.5 μm or less. The homogenized colostrum was then filtered through a filter membrane. The filtrate was filled into a 50 mL infusion bottle under nitrogen gas protection and sterilized in a rotary hot press at 121°C for 12 minutes to obtain an α-asarone injection emulsion containing α-asarone at a concentration of 1 mg / mL.

[0069] Preparation Example 5 Preparation of α-asarone oral emulsion The production method was the same as in Example 1. A pharmaceutically acceptable amount of antioxidants, such as vitamin E or pyrogallic acid esters, can be added to the oil phase, and a pharmaceutically acceptable amount of preservatives, such as ethylparaben, can be added to the oil phase. A pharmaceutically acceptable amount of flavoring, such as fragrant fruit juice syrup, can be added to the aqueous phase, and a pharmaceutically acceptable amount of preservatives, such as benzoic acid or sodium benzoate, can be added to the aqueous phase. Colostrum was also produced in the same manner. Subsequently, the colostrum was homogenized 1 to 3 times in a high-pressure homogenizer until the average particle size of the homogenized milk droplets became 10 μm or less. The homogenized colostrum was then filtered through a filter membrane. The filtrate was filled into appropriate pharmaceutical packages under nitrogen gas protection and subjected to fluidized bed steam sterilization at 100°C for 30 minutes or 121°C for 8 minutes, yielding an α-asarone oral emulsion.

[0070] Preparation Example 6 Preparation of α-asarone injectable emulsion 1.0-20.0 g of α-asarone and 50.0-200.0 g of soybean oil for injection were weighed and placed in a suitable container. Under nitrogen gas protection, the mixture was heated to 60-80°C and stirred to dissolve. Next, 12.0 g of egg yolk lecithin and 0.3 g of oleic acid were weighed and added to the container. The mixture was stirred and dissolved to prepare the oil phase. 22.0 g of glycerol was weighed and approximately 800 mL of water was weighed and heated to 60-80°C under nitrogen gas protection. The mixture was stirred and dissolved to prepare the aqueous phase. The oil phase was added to the aqueous phase and sheared at high speed for 5-15 minutes. Water was added to the mixture to make a total of 1000 mL to produce colostrum. The colostrum was homogenized 1 to 3 times using a high-pressure homogenizer until the average particle size of the homogenized milk droplets was 0.5 μm or less. The homogenized colostrum was then filtered through a filter membrane. The filtrate was filled into 2 mL, 5 mL, and 10 mL glass ampoules under nitrogen gas protection and sterilized in a rotary hot press at 121°C for 8 to 12 minutes to obtain α-asarone emulsion injections with α-asarone contents ranging from 1 mg / mL to 20 mg / mL.

[0071] Preparation Example 7 Preparation of α-asarone injection emulsion (also known as emulsion injection) Test materials: α-Asalone (2883-98-9, Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.) Soybean oil for injection (DD20200603, Shandong Ruisheng Pharmaceutical Excipients Co., Ltd.) Egg yolk lecithin (202008013, Shanghai Taiwei Pharmaceutical Co., Ltd.) Oleic acid (160907, Xi'an Libang Pharmaceutical Co., Ltd.) Glycerol (20191213, Zhejiang Suichang Huikang Pharmaceutical Co., Ltd.) Experimental steps: 10.0 g of α-asarone and 100.0 g of soybean oil for injection were weighed and placed in a suitable container. Under nitrogen gas protection, the mixture was heated to 80°C and stirred to dissolve. Next, 12.0 g of egg yolk lecithin and 0.3 g of oleic acid were weighed and added to the container, followed by stirring to dissolve the mixture. 22.0 g of glycerol was weighed, and approximately 800 mL of water was weighed and heated to 80°C under nitrogen gas protection, followed by stirring to dissolve the mixture. The oil phase was added to the aqueous phase, and the mixture was then sheared at 19,000 r / min for 10 minutes to disperse the oil phase into the aqueous phase. Water was then added to the container to make 1,000 mL of colostrum. The colostrum was then homogenized three times using a high-pressure homogenizer at a pressure of 1000 bar until the average particle size of the homogenized milk droplets was 0.5 μm or less. The homogenized colostrum was then filtered through a filter membrane. The filtrate was filled into 2 mL, 5 mL, and 10 mL glass ampoules under nitrogen gas protection and sterilized by rotary hot press at 121°C for 8 minutes to obtain an α-asarone emulsion injection with an α-asarone content of 10 mg / mL, batch number 20201228.

[0072] Preparation Example 8 Preparation of α-asarone oral emulsion 10.0 g of α-asarone and 100.0 g of medicated soybean oil were weighed and placed in a suitable container. Under nitrogen gas protection, the mixture was heated to 80°C and stirred to dissolve. Subsequently, 12.0 g of egg yolk lecithin, 0.3 g of oleic acid, 10.0 g of antioxidant vitamin E, and 2.0 g of ethylparaben were weighed and added to the container. The mixture was stirred and dissolved to prepare the oil phase. 22.0 g of glycerol was weighed and approximately 800 mL of water was weighed and heated to 80°C under nitrogen gas protection. The mixture was stirred and dissolved to prepare the aqueous phase. The oil phase was added to the aqueous phase and dispersed in the aqueous phase by high-speed shearing at 19,000 rpm for 10 minutes. Water was then added to the container to make 1,000 mL of colostrum. Subsequently, the colostrum was homogenized three times in a high-pressure homogenizer at a pressure of 1000 bar until the average particle size of the homogenized milk droplets was 0.5 μm or less. The homogenized colostrum was then filtered through a filter membrane. The filtrate was filled into 10 mL oral vials under nitrogen gas protection, and subjected to flow steam sterilization at 100°C for 30 minutes or rotary hot press sterilization at 121°C for 8 minutes to obtain an α-asarone oral emulsion with an α-asarone content of 10 mg / mL, the batch number of which is 20210105.

[0073] Efficacy Example 1: Short-term therapeutic effects of α-asarone on SAH and ICH rats Experimental materials: SPF grade SD rats, half male and half female, weighing 200-240 g, were purchased from Chengdu Dashuo Laboratory Animal Co., Ltd., Sichuan Province. The certificate number is SCXK(Sichuan)2020-030.

[0074] Collagenase VII was purchased from Sigma-Aldrich Company, USA (specification: 1.5KU, batch number: 0000111586).

[0075] The raw material α-asarone was purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd. (specification: 2 kg, batch number: 2883-98-9), and the emulsion injection was self-prepared with batch numbers 20201228 and 20210105.

[0076] Vinpocetine injection was purchased from Henan Runhong Pharmaceutical Co., Ltd. (specification: 10 mg: 2 mL, batch number: 1811283).

[0077] Nimodipine injection was purchased from Bayer Healthcare Company (specification: 10 mg: 50 mL, batch number: BXJC71).

[0078] Experimental grouping: Two hours after intravascular puncture or collagenase VII injection, the success of modeling was determined by Zea Longa score, and rats with successful modeling were randomly divided into groups for administration.

[0079] The rats were randomly divided into a sham operation group (Group P, administered with the same volume of saline as the high-dose emulsion injection group), a model group (Group S or I, administered with the same volume of blank emulsion as the high-dose emulsion injection group), a low-dose α-asarone emulsion injection group (prepared in Example 7, 7.5 mg / kg, Group L), a medium-dose α-asarone emulsion injection group (prepared in Example 7, 15 mg / kg, Group M), a high-dose α-asarone emulsion injection group (prepared in Example 7, 30 mg / kg, Group H), and The animals were divided into groups administered oral asarone emulsion (prepared in Example 8, 40 mg / kg, Group O), β-asarone emulsion injection group (prepared using the same method as in Example 7, prepared as an emulsion injection, concentration 10 mg / mL, dosage 20 mg / kg, Group B), vinpocetine injection group (commercially available, 2 mg / kg, Group V), and nimodipine injection group (commercially available, 1 mg / kg, Group N), with 12 animals in each group. Group N was administered intraperitoneally, while the other groups were administered via the tail vein.

[0080] 1.1 Construction of SAH by intravascular puncture Rats were fasted for 12 hours prior to surgery. Anesthesia was induced with 4% isoflurane and maintained with 2% isoflurane. The animals were placed in a supine position, and their body temperature was maintained at approximately 37°C. The neck skin was prepared, a midline incision was made, and the muscle and fascia were separated along the medial edge of the sternocleidomastoid muscle to expose the right side. The common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were bluntly separated, and the proximal end of the CCA, ICA, and ECA were pre-wired and pre-wired. The proximal end of the CCA and ECA were ligated, and the ICA was temporarily clamped with an arterial clip. A small hole was then drilled with a needle approximately 4 mm from the CCA to the bifurcation, and a puncture wire was inserted into the ICA via the CCA. The ICA arterial clip was then released, and the puncture wire was inserted intracranially. Resistance was felt when the tip of the catheter was approximately 18-19 mm from the bifurcation of the common carotid artery, indicating that the tip had reached the bifurcation of the anterior and middle cerebral arteries. After that, slight force was applied to extend the catheter approximately 2 mm, and the catheter penetrated the bifurcation of the anterior and middle cerebral arteries. The catheter was then completely removed, the ICA was ligated, and the wound was washed with saline and sutured. In the sham-operated group, the catheter was simply inserted and withdrawn when resistance was felt, without puncturing the bifurcation of the anterior and middle cerebral arteries. The remaining surgical procedures were the same as those in the experimental group. After waking from anesthesia, the animals were fed normally.

[0081] The severity of SAH was scored after completing the short-term neurological function score and euthanizing the rats. The score was based on the presence of subarachnoid hemorrhage in the basal cisterns and on the brain tissue surface. The basal cisterns were divided into six regions by the circle of Willis, which consisted of the basilar artery, anterior cerebral artery, internal carotid artery, posterior cerebral artery, and posterior communicating artery. Each region was scored from 0 to 3 points depending on the number of subarachnoid clots present. 0: no subarachnoid hemorrhage; 1: small amount of subarachnoid hemorrhage; 2: moderate clots, with the basilar artery identifiable; and 3: clots covering all arteries in that region. The six part scores were summed for a total of 18 points. Based on the final score, the severity of SAH hemorrhage was classified as follows: 0–7 points: mild subarachnoid hemorrhage; 8–12 points: moderate subarachnoid hemorrhage; and 13–18 points: severe subarachnoid hemorrhage. Moderate to severe SAH models were selected for statistical inclusion (score ≥ 8).

[0082] 1.2 Construction of an ICH model using collagenase injection Rats were fasted for 12 hours before surgery, and anesthesia was induced with 4% isoflurane and maintained with 2% isoflurane. The animals were kept in a prone position and their body temperature was maintained at approximately 37°C. The skin on the head was prepared, and a midline incision was made. According to the book Stereotaxic Mapping of the Rat Brain (George Paxinos, Charles Watson, Paxinos, Watson, & Zhuge Qi-Chan. Stereotaxic Mapping of the Rat Brain [M]. People's Health Publishing House, 2005), the right caudate nucleus of the rat was located using a stereotaxic instrument (the anterior fontanel was the origin, 3 mm to the right, 5.5 mm deep). After marking, a microsyringe needle was inserted into the caudate nucleus of the brain tissue using a drill hole in the skull. 1 μL of 0.5 U type VII collagenase was injected. The injection time was 5 minutes. After the injection was completed, the needle was not removed and held for 8 minutes. The needle was then slowly withdrawn, the skull hole was closed with bone wax, the skin was sutured, and the rat was returned to its cage. Rats in the sham-operated group received only sterile saline injections, with no other changes.

[0083] 1.3 Inclusion criteria for intracerebral hemorrhage models According to the Zea Longa neurological function score, rats were scored 2 hours after surgery after waking up from anesthesia, and rats with scores of 1 to 3 were included in the group.

[0084] 0 points: No neurological deficits, normal activity.

[0085] Score 1: Unable to fully extend opposite front leg.

[0086] 2 points: The animal rotates in circles as it crawls.

[0087] 3 points: The body falls to the hemiplegic side.

[0088] 4 points: Unable to walk spontaneously and loses consciousness.

[0089] 1.1 Short-term neurological deficit score Twenty-four hours after modeling, the rats' neurological function was comprehensively evaluated using the Garcia score and the Beam-balance test. The Garcia score criteria (Table 1) evaluate the rats' movement, sensation, crawling, and limb symmetry, with scores ranging from 3 to 18 points, with lower scores indicating more severe neurological damage. The Beam-balance test score criteria (Table 2) evaluate the rats' proprioception and physical coordination, with scores ranging from 0 to 6 points, with higher scores indicating more severe neurological damage. Scoring was performed independently by a blind person not involved in modeling or drug administration.

[0090] [Table 1]

[0091] [Table 2]

[0092] As shown in Table 3, 24 hours after surgery, the Garcia scores of the model groups (groups S and I) were significantly reduced (P<0.001) and the Beam-Balance scores were significantly increased (P<0.001) compared with the sham-operated group (group P). 24 hours after SAH or ICH, rats in the model groups exhibited significant neurological deficits. Intravenous administration of α-asarone (groups L, M, and H) and oral administration of α-asarone (group O) at different doses improved the Garcia scores and Beam-Balance scores to varying degrees, thereby ameliorating the neurological deficits caused by SAH or ICH. Among these, administration of group M had the most significant improvement (P<0.01). In the SAH model, the therapeutic effect of group M was superior to that of nimodipine (group N), a drug used to ameliorate vasospasm after subarachnoid hemorrhage, and vinpocetine (group V), a drug used to treat the sequelae of cerebral hemorrhage. In the ICH model, the therapeutic effects of Groups L and M were superior to Group V, and both were significantly superior to Group N. Conversely, the β-asarone-treated group (B) showed no significant improvement in neurological function in SAH and ICH model rats. Furthermore, there was no significant difference in hemorrhage scores between the model and treatment groups after brain extraction via cardiac perfusion in each SAH group, excluding differences in behavioral function caused by differences in the degree of modeling.

[0093] [Table 3] NOTE: Compared with the sham-operated group (P group), ### P<0.001, ## P<0.01, # P<0.05, compared with the model group (S or I group). ** P<0.01, * P<0.05, compared with the nimodipine group (N group). & P<0.05. Comparative data are shown as x±SD, and multigroup comparison analysis was performed using ANOVA with Tukey's post-hoc analysis.

[0094] Efficacy Example 2: α-Asarone reduces the incidence of secondary epilepsy in SAH rats The experimental materials, grouping, modeling method and administration method were the same as in Example 1, and the epileptic seizure status of rats in each group was observed within 24 hours after SAH in rats. The results are shown in Table 4. Compared with group P, group S had a significant increase in Racine score (P<0.001), while groups L, M, H and O could reduce Racine score to different degrees, with group M having the most significant effect of reduced administration (P<0.05). Therefore, α-asarone can significantly reduce the incidence of secondary epilepsy caused by SAH in rats.

[0095] [Table 4] Note: Compared with group P (administered saline), ### P<0.001, # P<0.05 compared with group S (administered blank emulsion). * P<0.05. Seizure grades are classified into six grades according to the severity of epileptic seizures based on the Racine criteria: Grade 0 is unresponsive or twitching arrest, Grade I is rhythmic mouth or facial twitching, Grade II is nodding or tail flicking, Grade III is single limb spasm, Grade IV is multiple limb spasm or tonic seizure, and Grade V is generalized tonic-clonic seizure. Grades I, II, and III are clonic seizures, and Grades IV and V are tonic seizures.

[0096] Efficacy Example 3: Long-term protective effect of α-asarone on SAH rats 3.1 Long-term survival rate of rats The experimental materials, grouping and modeling methods are the same as in Example 1. Two hours after SAH modeling, they were immediately administered according to the grouping administration method, and then continued to be administered for 14 days, once a day, and the survival status of the rats was observed and recorded for 14 days. The results are shown in Table 5. The mortality rate of group S was high at 53.8% within 24 hours, while the administration of groups M, H, O and N could significantly reduce the mortality rate of SAH rats within 24 hours and extend their survival time. That is, α-asarone can significantly reduce the 24-hour mortality rate of SAH rats and extend their survival time for 14 days.

[0097] [Table 5]

[0098] 3.2 Evaluation of long-term learning and memory function The Morris water maze was used to evaluate the long-term spatial perception and memory abilities of rats in each group. This was performed after the end of the survival observation period, i.e., 15–19 days after SAH. The water maze was a circular pool with a diameter of 150 cm and a depth of 60 cm. Before the experiment, warm water (24 ± 2°C) was added to a depth of 30 cm and dyed black using pigment. The pool was divided into four quadrants, and different labels were attached to the walls of the pools of different quadrants to distinguish them. A colorless, transparent platform measuring 10 cm in diameter and 28 cm high was placed in the center of one quadrant, submerged 2 cm in water. After the start of the experiment, rats were released into the designated quadrant according to the experimental guidelines. On days 1–4, rats were released into the water from four different quadrants in one experiment, with a 10-minute interval between trials between each round. If the rat found the platform within 60 seconds, it was allowed to stand on the platform for 10 seconds; if not, it was guided to the platform using a rod and allowed to stand on it for 10 seconds. On the fifth day, the platform was removed, and the rats were allowed to swim freely for 60 seconds. Data such as escape latency, swimming speed, and target quadrant exploration time were recorded using a computer tracking system (Noldus Ethovision, Tacoma, WA, USA).

[0099] As shown in Figure 1A, during the acquisition training period, the latency to find the platform was significantly longer in Groups S and N compared with Group P (P<0.001). The escape latency of Group M was significantly shorter than that of Groups S and N, and was not statistically different from Group P even on day 4. Figures 1B and 1C show that during spatial exploration, Groups S and N spent less time in the target quadrant compared with Group P, while Group M spent more time in the target quadrant, comparable to Group P. Furthermore, the swimming speed of Group M was significantly faster than Group S (P<0.05). As shown in Figure 1D, after long-term administration, both Groups S and N exhibited pallor and atrophy in the affected side of the brain tissue, but not Groups P and M. In conclusion, long-term administration of α-asarone not only significantly improved learning and memory function and promoted motor function recovery during the recovery period in SAH rats, but also attenuated brain tissue atrophy during the recovery period in SAH rats.

[0100] Efficacy Example 4: Study on the mechanism of action of α-asarone in antihemorrhagic stroke Test materials: Evans blue (C11891158, Shanghai Macklin Biochemical Co., Ltd.), Formamide (20190716, Tianjin BODI Chemical Co., Ltd.), Glutamate detection kit (20210525, Beijing Solarbio Science &Technology Co., Ltd.), GABA-Elisa kit (202101, Shanghai Jianglai Biology Co., Ltd.), DNase I (226F031, Beijing Solarbio Science&Technology Co., Ltd.), papain (111S022, Beijing Solarbio Science & Technology Co., Ltd.); Calcium ion fluorescent probe (20210313, Jiangsu Kaiji Biological Technology Co., Ltd.), Rhodamine 123 dye solution (119I033, Beijing Solarbio Science &Technology Co., Ltd.); Ice-cold centrifugation buffer (20210525, Beijing Solarbio Science & Technology Co., Ltd.).

[0101] Experimental steps and results: 4.1 Measurement of brain water content and blood-brain barrier permeability After completing the 24-hour short-term neurological function score, rats were deeply anesthetized by injecting 4% Evans blue solution (2.5 mL / kg) into the right tail vein. One hour later, the rats were deeply anesthetized and intracardially injected with 100 mL of saline. They were then rapidly decapitated and their brains were removed and immediately divided into left and right hemispheres, cerebellum, and brainstem. The left and right coronal hemispheres were divided into two halves, and each halve was weighed separately (wet weight) on a balance accurate to 0.1 mg. The samples were then dried in a 105°C oven for 24 hours and reweighed separately (dry weight). Brain water content was calculated as follows: brain water content = [(wet weight - dry weight) / wet weight] × 100%. The wet weight of the other parts of the brain tissue was weighed, then immersed in 10 volumes of pure formamide, incubated at 60°C for 48 hours, centrifuged at 25°C and 10,000 rpm / min for 30 minutes, the supernatant was aspirated and Evans blue dye was detected at 622 nm by UV spectrophotometry, and a standard curve for quantification was drawn, and the final results were expressed as Evans blue content per gram of brain tissue (μg / g).

[0102] The results are shown in Figure 2. Compared with the sham-operated group (P group), the cerebral hemisphere on the hemorrhage side of the model group (S or I group) had significantly increased brain water content, Evans blue exudation, and increased blood-brain barrier permeability 24 hours after surgery. Intravenous administration of α-asarone into the brain (M group) significantly reduced the water content of the brain tissue on the hemorrhage side of rats, alleviated cerebral edema, reduced Evans blue exudation, and improved blood-brain barrier permeability.

[0103] 4.2 Measurement of glutamic acid and GABA content Twelve to 24 hours after modeling, the rats were deeply anesthetized, decapitated, and their brains were removed. Approximately 60 to 120 mg of the cerebral cortex from the hemorrhagic side was collected, and 10 times the volume of ice-cold centrifugation buffer used for biochemical detection was added. The sample was homogenized in an ice bath for 10 minutes and centrifuged at 14,000 rpm / min at 4°C for 30 minutes. The supernatant was collected and the glutamate and GABA contents were detected according to the instructions of the glutamate content detection kit and rat GABA ELISA kit, respectively.

[0104] As shown in Figure 3, compared with group P, the cerebral hemisphere of rats in groups S and I had significantly increased glutamate and GABA levels 12 to 24 hours after surgery. However, administration of group M significantly reduced the glutamate and GABA levels in the brain tissue of rats with cerebral hemorrhage, counteracting glutamate excitotoxicity and restoring the balance of excitatory amino acids / inhibitory amino acids (EAA / IAA) in the brain, which is beneficial for improving motor function in rats.

[0105] 4.3 Ca 2+ Measurement of content or mitochondrial membrane potential Twelve to 24 hours after modeling, the animals were decapitated to remove the brain, and the cortex of the cerebral hemisphere surrounding the hematoma was collected. A single-cell suspension was immediately prepared by enzymatic digestion (papain: 2 mg / mL, DNase I: 0.05 mg / mL). The cell concentration was adjusted to 5 × 10 6 The cell suspension was adjusted to cells / mL, and 100 μL of the cell suspension was added to a final concentration of 5 μM Fluo-3 / AM staining solution or a final concentration of 10 μM Rhodamine 123 staining solution and incubated at 37°C for 45 minutes. The cells were washed twice with PBS (phosphate buffer, pH 7.2-7.4) and resuspended in 0.5 mL of PBS. 10,000 cells were detected using flow cytometry under conditions of an excitation wavelength of 506 nm and an emission wavelength of 526 nm. Fluo-3 and Rh123 mean fluorescence intensities were analyzed using Flowjo software.

[0106] The results, as shown in Figure 4, showed that compared with group P, groups S and I had significantly increased calcium ion content, elevated mitochondrial membrane potential, mitochondrial damage, and increased apoptosis 12 to 24 hours after surgery. However, administration of group M significantly reduced calcium ion content and stabilized mitochondrial membrane potential, thereby reducing neuronal apoptosis and necrosis.

[0107] Effect Example 5: Protective effect of α-asarone on oxyhemoglobin-damaged nerve cells Test materials: PC12 cell line was purchased from Wuhan Pronosai Life Sciences Co. Oxyhemoglobin (20210201, Beijing Solarbio Science & Technology Co., Ltd.), MTT (C12029690, Sigma-Aldrich, USA), DMEM high glucose medium (AG29301810, Hyclone, USA), fetal bovine serum (20010401, Gibco, USA); Penicillin-streptomycin solution (double antibody) (20201220, Hyclone, USA), PBS powder (WK173618-1, Beijing Zhongshan Jinqiao Biological Technology Co., Ltd.), DMSO(20201220, Beijing Solarbio Science&Technology Co., Ltd.) Experimental steps: Complete medium: DMEM high glucose medium, fetal bovine serum, and penicillin-streptomycin solution (double antibody) are mixed in a volume ratio of 90:9:1 and stored in a refrigerator at 4 °C.

[0108] Serum-free medium: DMEM high glucose medium and penicillin-streptomycin solution (double antibody) are mixed uniformly at a volume ratio of 99:1 and stored in a refrigerator at 4°C.

[0109] PC12 cells in the logarithmic growth phase cultured in complete medium were taken and 1 × 10 4 Cells were seeded into a 96-well plate at 100 μL / well, the edge wells filled with sterile PBS, and incubated at 37°C, 5% CO2 for 24 hours until the cells completely adhered to the wells. The supernatant was discarded, and oxyhemoglobin was added to final concentrations of 0 μM, 4 μM, 6 μM, 8 μM, and 10 μM, respectively, and incubated at 37°C, 5% CO2 for 24 hours. Next, 10 μL of 5 mg / mL MTT was added to each well, and the plate was incubated at 37°C, 5% CO2 for 4 hours. Discard the supernatant, add 100 µL / well of DMSO, and shake at 37 °C and 500 rpm for 15 minutes to completely dissolve the formazan. Use a microplate reader to measure the OD at 570 nm. To calculate the vitality of PC12 cells, use the formula: "abnormal cell proliferation fold = [(mean absorbance value of the experimental group - mean absorbance value of the zero-adjusted wells) / (mean absorbance value of the control group) - mean absorbance value of the zero-adjusted wells]." Under these experimental conditions, a maximum abnormal cell proliferation fold of 1.5 was determined to correspond to a concentration of 6 µM oxyhemoglobin. Therefore, this concentration was adopted as the preferred concentration for the oxyhemoglobin-induced oxidative stress injury model in preliminary studies and was used in the cellular pharmacodynamics experiments described below.

[0110] In addition, PC12 cells in the logarithmic growth phase cultured in complete medium were taken, and 1 × 10 4Cells were seeded into a 96-well plate at 100 μL / well, the edge wells filled with sterile PBS, and incubated at 37°C and 5% CO2 for 24 hours until the cells completely adhered to the wells. The supernatant was discarded, and 100 μL of α-asarone emulsion (prepared in Example 7, with final concentrations of α-asarone of 1 μM, 5 μM, 10 μM, 25 μM, and 50 μM, respectively) diluted with serum-free medium was added to each treatment group, except for the control and model groups. After 2 hours of incubation, oxyhemoglobin solution diluted with serum-free medium to a final concentration of 6 μM was added to each treatment group, and 20 μL / well was added at 37°C and 5% CO2 for further incubation. The control group received the same volume of serum-free medium alone. To the model group, 100 μL of blank emulsion (same as in Example 7 except that it did not contain α-asarone) diluted with serum-free medium and 20 μL of oxyhemoglobin solution diluted with serum-free medium to a final concentration of 6 μM were sequentially added. The remaining procedures for the control and model groups were the same as for the treatment groups. Next, 10 μL of 5 mg / mL MTT was added to each well and incubated at 37°C and 5% CO2 for 4 hours. The supernatant was discarded, and 100 μL of DMSO was added per well. The plates were shaken at 37°C and 500 rpm for 15 minutes to completely dissolve the formazan. The OD at 570 nm was detected using a microplate reader. The results are shown in Figure 5.

[0111] Experimental Results: As shown in Figure 5, compared with the control group, the addition of oxyhemoglobin caused abnormal proliferation of PC12 cells in the model group, with a significant increase in absorbance, a significant improvement in cell vitality, and an obvious oxidative stress response. Meanwhile, α-asarone at different concentrations significantly reduced the abnormal increase in cell vitality caused by oxyhemoglobin, indicating that it can significantly reduce the oxidative stress response induced by oxyhemoglobin (Figure 5).

[0112] Effect Example 6: Preliminary safety evaluation of α-asarone emulsion injection - Mouse bone marrow micronucleus experiment Experimental materials: 50 male Kunming mice of SPF grade, weighing 18 - 22 g, purchased from Chengdu Dashuo Laboratory Animal Co., Ltd., with the license number SCXK(Sichuan)2020 - 030). Cyclophosphamide for injection was purchased from Jiangsu Shengdi Pharmaceutical Co., Ltd. 1,4 - Piperazinediethanesulfonic acid (PIPES, 715H021, Beijing Solarbio Company), TritonX - 100(829I0210, Beijing Solarbio Company), Propidium Iodide (PI, 1024S043, Beijing Solarbio Company).

[0113] Grouping and administration of the experiment: The animals were randomly divided into five groups of 10 each, namely the blank control group (blank group, administered with a blank emulsion of the same volume as the high - dose α - asarone group), the cyclophosphamide group (CTX group, 40 mg / kg), the low - dose α - asarone emulsion injection group (manufactured from Production Example 7, 100 mg / kg / day, ASA - L group), the medium - dose α - asarone emulsion injection group (manufactured from Production Example 7, 150 mg / kg / day, ASA - M group), and the high - dose α - asarone emulsion injection group (manufactured from Production Example 7, 200 mg / kg / day, ASA - H group).

[0114] All drugs were injected via the tail vein. The positive control drug, cyclophosphamide (CTX), was injected once 24 hours before sampling. The other groups were administered continuously via the tail vein for 4 days. The mice were sacrificed by cervical dislocation 24 hours after the last administration. After separating the femurs from both sides, the femoral bone marrow cells were washed away with PBS, passed through a 300 - mesh nylon mesh to prepare a single - cell suspension, centrifuged at 1650 rpm for 5 minutes, resuspended in PBS, and after that, the cell concentration was adjusted to 5×10 6The cell concentration was adjusted to 100 μL / mL, and 100 μL of the cell suspension from each sample was carefully added to 400 μL of PIPES-PI solution (10 mL PIPES solution (concentration 3.5 mg / mL) + 0.5 mg PI + 0.01 mL Triton X-100 (concentration 0.1%)). The mixture was gently mixed and then incubated at 4°C for 30 minutes in the dark. The cells were then stained and detected by flow cytometry. The results are shown in Table 6. As shown in Table 6, PCE is polychromatic erythrocytes, MNPCE is polychromatic erythrocytes with micronuclei, and fMNPCE is the proportion of polychromatic erythrocytes containing micronuclei. This reflects the proportion of micronuclei in mouse bone marrow cells, with higher values ​​indicating greater genotoxicity.

[0115] As shown in Table 6, the micronucleus rate in the positive control drug cyclophosphamide group (CTX) was significantly increased compared to the blank emulsion group (P<0.01). There was no significant difference in the micronucleus rate between the blank emulsion group and each dose of α-asarone emulsion injection. Compared to the CTX group, the micronucleus rate was significantly lower, with statistical differences (ASA-L: P<0.01, ASA-M: P<0.05, ASA-H: P<0.05).

[0116] The above-mentioned toxicological study on chromosomal damage in mouse hematopoietic cells showed that intravenous administration of α-asarone emulsion injection up to 200mg / kg did not significantly change the micronucleus rate in mouse bone marrow cells. Considering the effective dose for the treatment of hemorrhagic stroke, the safety of this drug is expected to be good.

[0117] [Table 6] Note: Compared to the blank group, ## P<0.01 compared with the CTX group. ** P<0.01, * P<0.05.

[0118] In summary, the results of in vitro and in vivo pharmacodynamic studies showed that α-asarone significantly improved short-term neurobehavioral and long-term learning and memory functions in rats with hemorrhagic stroke, reduced the incidence and mortality of secondary epilepsy in SAH rats, alleviated cerebral edema, improved blood-brain barrier permeability, prevented or alleviated brain tissue atrophy during the recovery period, antagonized glutamate excitotoxicity, restored GABA levels, restored the excitatory amino acid / inhibitory amino acid (EAA / IAA) balance in the brain, and reduced Ca. 2+ It has been shown that α-asarone can reduce the influx of β-amyloid, stabilize mitochondrial membrane potential, reduce neuronal apoptosis, alleviate oxidative stress, and exert neuroprotective effects. Therefore, α-asarone is expected to be a promising drug for the treatment of hemorrhagic stroke.

[0119] Although specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely examples and that various modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. Use of a compound of formula I in the manufacture of a medicament for treating hemorrhagic stroke resulting from at least one of intracerebral hemorrhage and subarachnoid hemorrhage, wherein the compound of formula I is the only active ingredient of the medicament having activity in treating hemorrhagic stroke. 【Chemistry 1】

2. The use according to claim 1, characterized in that the drug is used for at least one of improving neurological or motor function damage caused by ICH or SAH, improving acute brain tissue edema or blood-brain barrier dysfunction caused by ICH or SAH, reducing acute mortality due to hemorrhagic stroke, extending survival time, improving long-term learning and memory dysfunction caused by hemorrhagic stroke, and preventing or alleviating brain tissue atrophy during the recovery period from hemorrhagic stroke.

3. 2. The use according to claim 1, characterized in that the drug contains pharmaceutical auxiliaries.

4. The use according to claim 3, wherein the total weight ratio of the compound of formula I to pharmaceutical auxiliaries is 1:20-1000.

5. The use according to claim 1, characterized in that when the medicament is used to treat people suffering from hemorrhagic stroke, the daily dosage range of the compound of formula I of the medicament is 0.15 mg to 5.0 mg / kg body weight.

6. The use of claim 5, wherein the daily dosage range of the compound of formula I of the medicament is 0.3 mg to 3.0 mg / kg body weight when the medicament is used to treat a person suffering from hemorrhagic stroke.

7. The use according to claim 1, characterized in that the drug is administered by injection or oral administration.

8. The use according to claim 1, characterized in that the drug is an emulsion.

9. containing a compound of formula I and pharmaceutical auxiliaries, A pharmaceutical composition for use in the treatment of hemorrhagic stroke, characterized in that the compound of formula I is the only active ingredient having activity in treating hemorrhagic stroke. 【Chemistry 2】

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